Figure 6B Representative pulldown - experiment and quantification of 6His - tagged gephyrin and G375D using immobilized α3 - peptide . 6His - tagged gephyrin variants were expressed in E . coli cells , purified to homogeneity and incubated with α3 - peptides immobilized to NeutrAvidin beads . n = 3 , normalized to 6His - gephyrin . 38 . 9 ± 1 . 5 % G375D were pulled down . C Representative SPR sensograms of 6His - gephyrin and 6His - G375D binding to immobilized GABAAR α3 - peptide . Response units are plotted against time . Injected gephyrin concentrations were 0 . 5 , 2 , 10 , 25 , 40 µM . D SPR binding isotherms of a representative experiment as in C . Calculated KD values of 3 . 2 and 10 µM for gephyrin and G375D , respectively , are indicated by vertical lines . The experiment was repeated three times with two independently purified protein batches and two sensor chips . E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß - subunit ( GlyR - ßL , residue 378 - 426 ) and 6His - tagged gephyrin or G375D . The GlyR - ßL - gephyrin binding data ( black ) were fitted in a two - side model , whereas G375D binding could only be fitted to a single binding site . Dissociation constants KD and stoichiometry ( N = number of binding sites ) are given for each binding component . n = 5 from two independently purified protein batches . Results are expressed as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.


Figure 7A Biosynthesis pathway of Moco with all stable intermediates . Gephyrin G - and E - domain catalyze the last two steps as depicted . B In vitro Moco - synthesis assay using 150 pmol purified 6His - tagged gephyrins . D580A is an activity - deficient gephyrin mutation previously identified in Moco - deficient patients ( Reiss et al . , 2011 ) . Assays without the addition of gephyrin ( - gephyrin ) or molybdenum ( - Mo ) served as internal controls of the assay ( see also ( Belaidi Schwarz , 2013 ) ) . C Experiment as in B but with pre - incubation of 6His - gephyrin / G375D in a 1 : 1 ratio to simulate the heterozygous mutation . n = at least 3 with two independently purified protein batches . Results are expressed as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.


figf1 ( b ) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy . HeLa cells transfected with HA - Parkin were treated with CCCP for the indicated times . Mitochondria were stained by anti - TOM20 ( pseudo coloured ; blue ) and a ΔΨm dependent MitoTracker ( red ) . Parkin was stained with anti - HA ( green ) . Without treatment , mitochondria are intact and stained by both mitochondrial markers , whereas Parkin is equally distributed in the cytoplasm . After 2 h of CCCP treatment , mitochondria are depolarized as shown by the loss of MitoTracker staining . Parkin completely translocates to mitochondria clustering at perinuclear regions . After 24 h of CCCP treatment , massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker . Only Parkin - positive cells show mitochondrial clustering and clearance , in contrast to adjacent untransfected cells . Scale bars , 10 μm . ( c ) Pathogenic Parkin mutations abrogate mitochondrial translocation and / or clearance . HeLa cells transfected with Flag - Parkin wild - type or pathogenic mutants were treated with CCCP and stained with anti - Flag ( green ) , anti - TOM20 ( red ) and Hoechst33342 ( blue ) . Representative merged images are shown . Scale bars , 10 μm . Immunostaining of cells transfected with non - functional cysteine mutants and functional Parkin variants is shown in Supplementary Information , Fig . S1a and S1b , respectively . ( d , e ) Based on the mitochondrial effects , Parkin mutants can be classified into functional and non - functional classes . ( d ) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment , as defined by complete colocalization of Parkin with the mitochondrial marker ( n = 3 ) . ( e ) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment , as shown by complete loss of mitochondrial staining ( n = 3 ) . Data represent the mean ± s . d . , * P ≤ 0 . 05 , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with wild - type . WT , wild - type .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively.(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.


figf2Stable Parkin wild - type or pathogenic mutants expressing SH - SY5Y cell lines were induced with doxycycline ( DOX ) for 72 h and treated with CCCP for the indicated times . Cells were stained with anti - Parkin ( green ) , anti - TOM20 ( red ) and Hoechst33342 ( blue ) . ( a - b ) CCCP treatment of stable SH - SY5Y cells for 0 h . Unstimulated control cells ( - DOX ) show relatively low levels of endogenous Parkin , compared to cells with induced expression of Parkin wild - type or K161N , R275W and G430D mutants ( + DOX ) . Immunostaining ( a ) and western blot analysis ( b ) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones . β - Actin served as a loading control . ( c , d ) After 2 h of CCCP treatment , massive colocalization of wild - type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls . Similarly , Parkin K161N and R275W mutants translocate to mitochondria , whereas G430D does not . ( e , f ) After 24 h of CCCP treatment , induced wild - type Parkin - expressing cells show full clearance of damaged mitochondria . In contrast , the pathogenic mutants analysed fail to execute mitophagy and , therefore , damaged mitochondria remain uncleared . Representative images of three independent experiments are shown . Scale bars , 10 μm ( a , c and e ) . ( d , f ) Quantifications of the observed mitochondrial effects in Parkin - expressing SH - SY5Y cells ( n = 3 ) . Data represent the mean ± s . d . , * * * P ≤ 0 . 0005 compared with induced wild - type Parkin . WT , wild - type .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control.(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not.(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.


figf3 ( b ) Co - immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization . Endogenous Parkin and PINK1 were co - immunoprecipitated from neuronal SH - SY5Y cells after CCCP treatment with specific antibodies . ( c ) Western blot of PINK1 siRNA - transfected HeLa cells used in d - h demonstrates the knockdown efficiency and specificity of PINK1 silencing . d - h ) PINK1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , and mitochondrial clearance after 24 h , of CCCP treatment . This can be rescued by re - transfection with wild - type PINK1 , but not with a kinase - dead mutant or a PINK1 variant lacking the MTS . Control siRNA - or PINK1 siRNA - silenced HeLa cells were transfected with EGFP - Parkin wild - type and PINK1 - V5 wild - type , 3 × KD or ΔMTS mutants . Use of appropriate empty vector controls is depicted . Cells were treated with CCCP and stained with anti - TOM20 ( pseudo coloured ; blue ) and anti - V5 ( red ) to visualize mitochondrial morphology and re - transfected PINK1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells are surrounded by a line . Representative images of four independent experiments are shown . Scale bars , 10 μm . ( e ) Wild - type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed ( n = 4 ) . ( g ) Quantification of results from cells in e . ( f ) Uncleared mitochondria were analysed after 24 h of CCCP treatment ( n = 4 ) . ( h ) Quantification of results from cells in f . Data represent the mean ± s . d . , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with control siRNA - transfected cells . Immunostaining of control siRNA - transfected cells at all analysed time points is shown in Supplementary Information , Fig . S2 . WT , wild - type .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies.(c) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing.d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm.(e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e.(f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.


figf4a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa cells were transfected with Flag - Parkin wild - type or pathogenic mutants . Cells were treated with CCCP before immunostaining of Parkin with anti - Flag ( green ) and endogenous poly - ubiquitin with a specific antibody FK1 ( red ) . ( a ) In contrast to pathogenic mutations , wild - type Parkin shows colocalization with poly - ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria . ( b ) After 24 h of CCCP treatment , K161N and R275W Parkin mutants colocalize with both poly - ubiquitin and mitochondria . In contrast , translocation - deficient Parkin mutants K211N , T240R and G430D lack the mitochondria - associated poly - ubiquitin signal . Representative images are shown . Scale bars , 10 μm . Complementary Parkin of untreated cells and quantification of colocalization of Parkin , mitochondria and poly - ubiquitin upon CCCP treatment are shown in Supplementary Information , Fig . S3a , b and c , respectively . ( c , d ) Parkin - directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63 . HeLa cells were transfected with EGFP - Parkin wild - type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti - HtrA2 / Omi , as a mitochondrial marker ( pseudo coloured ; blue ) and anti - His ( red ) to detect ectopic ubiquitin . Parkin was visualized by its fusion to EGFP ( green ) . ( c ) Note that only wild - type , K27 and K63 6 × His - tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP - Parkin after 6 h of CCCP treatment . All other ubiquitin mutants interfere with Parkin translocation to mitochondria . ( d ) Only co - expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6 × His - tagged wild - type ubiquitin after 24 h of CCCP treatment . Representative images are shown . Scale bars , 10 μm . Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information , Fig . S4a and b , c , respectively . WT , wild - type ; PolyUb , poly - ubiquitin .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively.(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.


figf5 ( a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa cells were transfected with Flag - Parkin wild - type or pathogenic mutants . Cells were treated with CCCP for the indicated times before immunostaining with anti - Flag ( green ) and anti - p62 ( red ) . ( a ) In contrast to the pathogenic mutants , wild - type Parkin colocalizes with p62 at the site of clustered mitochondria , after 6 h of CCCP treatment . ( b ) After 24 h of CCCP treatment , wild - type Parkin - transfected cells show complete loss of mitochondria , whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria . In contrast , translocation - compromised mutants K211N , T240R and G430D , lacking the mitochondria - associated poly - ubiquitin signal ( Fig . 4b ) , did not colocalize with p62 . Representative images of four independent experiments are shown . Data from untreated control cells and quantification of colocalization of Parkin , mitochondria and p62 upon CCCP treatment are shown in Supplementary Information , Fig . 5a and b , c , respectively . ( c , d ) Control siRNA - or p62 siRNA - silenced HeLa cells were transfected with EGFP - Parkin wild - type . Cells were treated with CCCP for the indicated times and stained with anti - TOM20 ( pseudo coloured ; blue ) to visualize mitochondrial morphology and anti - p62 ( red ) to monitor silencing efficiency . Parkin wild - type was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells with cleared mitochondria are surrounded by a line . ( ( c - f ) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment ( c - e ) , but completely blocks final clearance after 24 h ( c , d , f ) . ( e , f ) Relative parkin translocation ( e ) and mitochondrial clearance ( f ) after p62 knockdown and CCCP treatment ( n = 4 ) . Data represent the mean ± s . d . , * * * P ≤ 0 . 0005 compared with control siRNA - transfected cells . Representative images of four independent experiments are shown . Scale bars , 10 μm . Immunostainings of control cells are shown in Supplementary Information , Fig . S5d , e . ( g ) Western blot of siRNA - transfected HeLa cells used in c - f demonstrates the efficiency of p62 silencing . β - Actin probing served as a loading control . WT , wild - type .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively.(c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. ((c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e.(g) Western blot of siRNA-transfected HeLa cells used in c-f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.


figf6 ( a ) Time - course experiment of HeLa cells transfected with Flag - Parkin wild - type and treated with CCCP for the indicated periods . Total lysates were prepared , and western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 and anti - VDAC1 antibodies . β - Actin probing served as a loading control . All of the proteins detected are endogenously expressed , except Flag - Parkin . ( b ) Pathogenic Parkin mutants fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . HeLa cells were transfected with empty vector ( - ) , Flag - Parkin wild - type or pathogenic mutants along with 6 × His - tagged ubiquitin wild - type for 24 h . Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer . Denaturing conditions of Ni - NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis - tagged ubiquitin . Western blots of protein lysates and purified Ni - NTA agarose elutions were probed with anti - VDAC1 , anti - TOM20 and anti - Parkin antibodies . Modification of VDAC1 was observed in cells expressing Parkin wild - type and the functional mutant G328E , whereas non - functional mutants did not have this effect . ( ( c ) In response to CCCP treatment , Parkin catalyses predominantly K27 - linked poly - ubiquitin chains on endogenous VDAC1 and is itself strongly poly - ubiquitylated . HeLa cells were transfected with an empty vector ( - ) or Flag - Parkin wild - type along with 6 × His - tagged ubiquitin variants for 24 h . Treatment of cells , preparation of lysates and Ni - NTA purification were performed as described in c and probed for Parkin , VDAC1 and β - actin . WT , wild - type ; Ub , ubiquitin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin.(b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. ((c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.


figf7 ( a ) Western blot analysis of parental SH - SY5Y cells treated with CCCP for the indicated times . Time - course experiments of CCCP - treated neuronal cells reveal increasing modifications of VDAC1 over time . Total lysates were prepared , and western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 and anti - VDAC1 antibodies . All proteins represent endogenous levels . ( b ) Immunoprecipitations of Parkin and VDAC1 from parental SH - SY5Y cells confirm ubiquitylation of both proteins . CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time . Using specific antibodies , Parkin and VDAC1 were immunoprecipitated from lysates of parental SH - SY5Y cells at different time points , western blotted and probed with anti - ubiquitin , anti - Parkin and anti - VDAC1 antibodies . ( c ) Pathogenic Parkin mutations fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . Western blot analysis of a time - course experiment with inducible Parkin SH - SY5Y cells after CCCP treatment . Total lysates were prepared , and western blots were probed with specific anti - Parkin and anti - VDAC1 antibodies . β - Actin probing served as a loading control in a - c . WT , wild - type ; Ub , ubiquitin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels.(b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies.(c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.


figf8VDAC1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , and mitochondrial clearance after 24 h , of CCCP treatment . Both effects can be rescued by re - transfection with wild - type VDAC1 . ( a ) Immunofluorescence and ( b ) western blot of siRNA - transfected HeLa cells used in c - g demonstrate partial silencing of endogenous VDAC1 . ( a ) Silenced cells were stained with anti - HtrA2 / Omi , a mitochondrial marker ( pseudo coloured ; blue ) , and anti - VDAC1 ( red ) antibodies . ( b ) Lysates from silenced cells were subjected to western blot analysis and probed with an anti - VDAC1 antibody , while TOM20 and β - actin probing served as loading controls . ( c - e ) Control siRNA - or VDAC1 siRNA - silenced HeLa cells were transfected with the indicated combinations of EGFP - Parkin wild - type , VDAC1 - Flag wild - type or appropriate empty vector controls . Cells were treated with CCCP for 2 h and 24 h and stained with anti - HtrA2 / Omi ( pseudo coloured ; blue ) and anti - Flag ( red ) to visualize mitochondrial morphology and re - transfected VDAC1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells are surrounded by a line . Representative pictures of three independent experiments are shown . Scale bars , 10 μm . ( f , g ) Quantification of d ( f ) and e ( g ) . Parkin wild - type colocalization with impaired mitochondria after 2 h ( f ) and uncleared mitochondria after 24 h ( g ) of CCCP treatment ( n = 3 ) . Data represent the mean ± s . d . , * P ≤ 0 . 05 compared with control siRNA - transfected or with VDAC1 - Flag wild - type - transfected cells .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c-g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls.(c-e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP-Parkin wild-type, VDAC1-Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm.(f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1-Flag wild-type-transfected cells.


Figure 1 ( A ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : EtOH : ethanol ; HAM : hydroxylamine ; 2 - BP : 2 - bromopalmitate ( B ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : FL : full length ; N : N - terminal domain ; C : C - terminal domain ( C ) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264 . 7 cells . Data information : PA : palmitic acid ; 17 - ODYA : 17 - octadecanoic acid . ( D and E ) In vitro palmitoylation assay for the indicated proteins . NBD - Palm - CoA : ( N - [ ( 7 - nitro - 2 - 1 , 3 - benzoxadiazol - 4 - yl ) - methyl ] amino ) palmitoyl - CoA . The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining . ( F ) LC - MS / MS analysis of palmitoylated peptides of cGAS . ( G ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : HAM : hydroxylamine ( H - I ) THP - 1 cells were treated with DMSO or 2 - BP ( 50 μM ) ( H ) or palmitic acid ( 100 μM ) ( I ) for 12 h . Six hours after transfection with HT - DNA ( 2 μg / mL ) , cGAMP was extracted and quantified by cGAMP ELISA . Data information : 2 - BP : 2 - bromopalmitate Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 ( J ) THP - 1 cells were treated with palmitic acid ( 0 , 50 μM , or 100 μM ) for 12 h before a cGAMP bioassay . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h and transfected with HT - DNA ( 2 μg / mL ) for 6 h before RT - qPCR analysis of Ifna4 ( K ) , IFNb1 ( L ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h and transfected with HT - DNA ( 2 μg / mL ) for 6 h before RT - qPCR analysis of , Cxcl10 ( M ) and Rantes ( N ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 ( O ) L929 cells were treated with BSA and palmitic acid ( 100 μM ) and transfected with HT - DNA ( 2 μg / mL ) for the indicated times before immunoblotting analysis with the indicated antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: EtOH: ethanol; HAM: hydroxylamine; 2-BP: 2-bromopalmitate(B) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: FL: full length; N: N-terminal domain; C: C-terminal domain(C) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264.7 cells. Data information: PA: palmitic acid; 17-ODYA: 17-octadecanoic acid.(D and E) In vitro palmitoylation assay for the indicated proteins. NBD-Palm-CoA: (N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA. The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining.(F) LC-MS/MS analysis of palmitoylated peptides of cGAS.(G) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: HAM: hydroxylamine(H-I) THP-1 cells were treated with DMSO or 2-BP (50 μM) (H) or palmitic acid (100 μM) (I) for 12 h. Six hours after transfection with HT-DNA (2 μg/mL), cGAMP was extracted and quantified by cGAMP ELISA. Data information: 2-BP: 2-bromopalmitate Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001(J) THP-1 cells were treated with palmitic acid (0, 50 μM, or 100 μM) for 12 h before a cGAMP bioassay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of Ifna4 (K), IFNb1 (L) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of , Cxcl10 (M) and Rantes (N) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001(O) L929 cells were treated with BSA and palmitic acid (100 μM) and transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies.


Figure 2 ( A ) Acyl - RAC assay of HEK293T cells transfected with Flag - cGAS 24 h after transfection with the indicated shRNA . ( B ) Quantification of palmitoylation levels of Flag - cGAS in ( A ) . Data information : ( C ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . HAM : hydroxylamine . ( D ) HEK293T cells were transfected with HA - cGAS ( WT or C474S mutant ) ( 200 ng ) , Flag - STING ( 200 ng ) and Myc - ZDHHC18 ( 200 ng ) expression plasmids for 24 h before luciferase reporter assays . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 ( D ( E and F ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; FL : full length ( H ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA - cGAS and Flag - ZDHHC18 truncations or GFP for 24 h . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; FL : full length ( J ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA - ZDHHC18 and Flag - cGAS truncates or GFP for 24 h . Data information : WCL : whole - cell lysate ( K ) Immunofluorescence analysis of HA - cGAS ( green ) and Flag - ZDHHC18 ( red ) in HT - DNA - stimulated ( or not ) HeLa cells . Scale bars : 7 μm . ( L ) Colocalization ( merged volume of total cGAS signal ) of cGAS and ZDHHC18 in ( K ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 L ) . ( M - N ) Comparison of the binding free energy ( M ) and binding energy decomposition of a per - residue to the binding affinity between ZDHHC18 and cGAS ( N ) in different complexes .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Acyl-RAC assay of HEK293T cells transfected with Flag-cGAS 24 h after transfection with the indicated shRNA. (B) Quantification of palmitoylation levels of Flag-cGAS in (A). Data information:(C) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine.(D) HEK293T cells were transfected with HA-cGAS (WT or C474S mutant) (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (200 ng) expression plasmids for 24 h before luciferase reporter assays. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 (D(E and F) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-cGAS and Flag-ZDHHC18 truncations or GFP for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length(J) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-ZDHHC18 and Flag-cGAS truncates or GFP for 24 h. Data information: WCL: whole-cell lysate(K) Immunofluorescence analysis of HA-cGAS (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa cells. Scale bars: 7 μm. (L) Colocalization (merged volume of total cGAS signal) of cGAS and ZDHHC18 in (K). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 L).(M-N) Comparison of the binding free energy (M) and binding energy decomposition of a per-residue to the binding affinity between ZDHHC18 and cGAS (N) in different complexes.


Figure 3 ( A ) Immunofluorescence analysis of GFP - cGAS , the nucleus ( DAPI ) , the ER ( calnexin ) , the Golgi apparatus ( GM130 ) , and early endosomes ( EEA1 ) in HeLa cells . 2 - BP : 2 - bromopalmitate ; ISD : IFN - stimulatory DNA . Arrows indicate cGAS puncta . Scale bars : 10 μm . ( F HEK293T cells were transfected with the indicated plasmids . Biotin - conjugated ISD was transfected into cells 6 h before harvesting . Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation . Data are representative of at least two independent experiments . G ) HEK293T cells were transfected with the indicated plasmids . Biotin - conjugated ISD was transfected into cells 6 h before harvesting . Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown . Data information : WCL : whole - cell lysate ; Data are representative of at least two independent experiments . ( H ) Confocal microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 − / − ) transfected with FITC - conjugated ISD for 6 h ( left ) . Scale bar : 20 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Immunofluorescence analysis of GFP-cGAS, the nucleus (DAPI), the ER (calnexin), the Golgi apparatus (GM130), and early endosomes (EEA1) in HeLa cells. 2-BP: 2-bromopalmitate; ISD: IFN-stimulatory DNA. Arrows indicate cGAS puncta. Scale bars: 10 μm.(F HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; IP: immunoprecipitation. Data are representative of at least two independent experiments.G) HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; Data are representative of at least two independent experiments.(H) Confocal microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18−/−) transfected with FITC-conjugated ISD for 6 h (left). Scale bar: 20 μm.


Figure 4 ( C ) Cross - correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins . ( D ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with plasmids encoding Flag - cGAS , HA - cGAS , and Myc - ZDHHC18 ( WT or CS mutant ) for 24 h . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; Data are representative of at least two independent experiments . ( E ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids followed by the addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments . ( F ) FRET assay of HeLa cells transfected with the indicated plasmids with the addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Scale bar : 8 μm . ( G ) FRET efficiency of cGAS - GFP and cGAS - mCherry in ( F ) . Data information : 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 ( G ( H ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with Flag - cGAS and HA - cGAS ( WT , C405S , C409S and C474S ) expression plasmids . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation Data are representative of at least two independent experiments . ( I ) FRET assay of HeLa cells transfected with the indicated plasmids . Scale bar : 8 μm . ( J ) FRET efficiency of cGAS - GFP and cGAS - mCherry in ( I ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 J ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C) Cross-correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins.(D) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding Flag-cGAS, HA-cGAS, and Myc-ZDHHC18 (WT or CS mutant) for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; Data are representative of at least two independent experiments.(E) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids followed by the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Data information: WCL: whole-cell lysate; IP: immunoprecipitation; 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments.(F) FRET assay of HeLa cells transfected with the indicated plasmids with the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Scale bar: 8 μm. (G) FRET efficiency of cGAS-GFP and cGAS-mCherry in (F). Data information: 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 (G(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with Flag-cGAS and HA-cGAS (WT, C405S, C409S and C474S) expression plasmids. Data information: WCL: whole-cell lysate; IP: immunoprecipitation Data are representative of at least two independent experiments.(I) FRET assay of HeLa cells transfected with the indicated plasmids. Scale bar: 8 μm. (J) FRET efficiency of cGAS-GFP and cGAS-mCherry in (I). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 J).


Figure 5 ( A ) HEK293T cells ( 5 × 105 ) were transfected with HA - cGAS ( 200 ng ) , Flag - STING ( 200 ng ) and Myc - ZDHHC18 ( WT or CS mutant ) expression plasmids ( 0 , 50 , 100 , or 200 ng ) for 24 h before luciferase reporter assays were conducted . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ( A ( B ) HEK293T cells ( 1 × 106 ) were transfected with HA - cGAS ( 500 ng ) , Flag - STING ( 500 ng ) and Myc - ZDHHC18 ( 500 ng ) expression plasmids for 24 h . TBK1 phosphorylation was detected by immunoblotting . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in the DHHC motif of ZDHHC18 ( C and D ) HEK293T cells were transfected with the indicated plasmids , and the amount of cGAMP in the lysates was quantified by LC - MS / MS . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in the DHHC motif of ZDHHC18 ; LC - MS / MS : liquid chromatography - tandem mass spectrometry ; L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT - qPCR ( E ) results . Data information : NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting ( F ) results . Data information : NC : negative control . L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before RT - qPCR analysis of Ifna4 ( G ) , expression . Data information : HT - DNA : herring testis DNA ; NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before RT - qPCR analysis of IFNb1 ( H ) , and Cxcl10 ( I ) expression . Data information : HT - DNA : herring testis DNA ; NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ( J ) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before immunoblotting analysis with the indicated antibodies . Data information : HT - DNA : herring testis DNA ; NC : negative control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) HEK293T cells (5×105) were transfected with HA-cGAS (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (WT or CS mutant) expression plasmids (0, 50, 100, or 200 ng) for 24 h before luciferase reporter assays were conducted. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (A(B) HEK293T cells (1×106) were transfected with HA-cGAS (500 ng), Flag-STING (500 ng) and Myc-ZDHHC18 (500 ng) expression plasmids for 24 h. TBK1 phosphorylation was detected by immunoblotting. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18(C and D) HEK293T cells were transfected with the indicated plasmids, and the amount of cGAMP in the lysates was quantified by LC-MS/MS. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18; LC-MS/MS: liquid chromatography-tandem mass spectrometry;L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) results. Data information: NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control.L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of Ifna4 (G), expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of IFNb1 (H), and Cxcl10 (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001(J) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.


Figure 6 ( A ) Survival analysis of Zdhhc18 + / + and Zdhhc18 - / - mice ( n = 8 each , 6 - 8 weeks old ) intranasally injected with VACV ( 5 × 107 PFU per mouse ) and monitored daily for survival for 13 days using Kaplan - Meier curves and a log - rank ( Mantel - Cox ) test . * * * , P ˂ 0 . 001 . ( B ) Representative photomicrographs of H & E - stained liver and kidney tissue sections from mice ( Zdhhc18 + / + or Zdhhc18 - / - ) intravenously injected with HSV - 1 - FS ( 5 × 107 PFU per mouse ) or not after 2 days . Scale bars : 100 μm ( liver ) , 200 μm ( kidney ) . Data information : PFU : plaque - forming units ; H & E : hematoxylin - eosin . Data are representative of at least two independent experiments . ( C BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments . D ) BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001 ( D ( E Frequency of GFP + cells among infected cells ( E ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001F ) Microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) infected with GFP - HSV - 1 ( MOI = 1 ) for 10 h . Scale bar : 200 μm . Data information : Data are representative of at least two independent experiments . ( G ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before immunoblot analysis . ( H and I ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of Cxcl10 ( H ) and Ifnb1 ( I ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001 ( J ) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA ( cocktail ) for 48 h and then infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of IFNB1 expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Survival analysis of Zdhhc18+/+ and Zdhhc18-/- mice (n = 8 each, 6-8 weeks old) intranasally injected with VACV (5 × 107 PFU per mouse) and monitored daily for survival for 13 days using Kaplan-Meier curves and a log-rank (Mantel-Cox) test. ***, P˂0.001.(B) Representative photomicrographs of H&E-stained liver and kidney tissue sections from mice (Zdhhc18+/+ or Zdhhc18-/-) intravenously injected with HSV-1-FS (5 × 107 PFU per mouse) or not after 2 days. Scale bars: 100 μm (liver), 200 μm (kidney). Data information: PFU: plaque-forming units; H&E: hematoxylin-eosin. Data are representative of at least two independent experiments.(C BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments.D) BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001 (D(E Frequency of GFP+ cells among infected cells (E). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Data information: Data are representative of at least two independent experiments.(G) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before immunoblot analysis.(H and I) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before RT-qPCR analysis of Cxcl10 (H) and Ifnb1 (I) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001(J) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA (cocktail) for 48 h and then infected with VACV (1:200) for the indicated times before RT-qPCR analysis of IFNB1 expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


Figure 1 ( C ) Quantitative real time PCR analysis showing head enrichment of dEsyt ; the X - axis indicates the wild type tissues from which RNA was isolated and Y - axis indicates the dEsyt transcript level expression normalized to the loading control ( RP49 - Ribosomal protein 49 ) , n = 3 replicates ( biological ) . ( D ) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD . Y - axis indicates the dEsyt transcript level expression normalized to the loading control ( RP49 - Ribosomal protein 49 ) , n = 3 replicates ( biological ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) Quantitative real time PCR analysis showing head enrichment of dEsyt; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).(D) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD. Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


Figure 2 ( A ) Representative ERG trace from dark reared 0 - 1 day old flies of the indicated genotype stimulated by 2s flash of green light . The duration of the stimulating light is shown . Y - axis shows the amplitude of the ERG response . X - axis shows the duration of the recording . ( B ) Graph showing ERG amplitude normalized to body weight . Y - axis shows the ratio of ERG amplitude of individual flies to their body weight . X - axis indicates genotypes . Each data point represents an individual fly tested . Error bar represents s . e . m . ( C ) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH - GFP in the one day old flies of the indicated genotypes ( n = 10 ) . ( D ) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors . The fluorescence of the PH - GFP probe is depicted . The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light ( a - f ) used for image capture depicted . Representative deep pseudopupil images acquired at specified time points are depicted . Genotypes as indicated . ( E ) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time , X - axis represents the genotypes , Y - axis represent intensity normalised to the intensity of the first image . ( n = 10 biological replicates ) . Error bars represent s . e . m . ( F ) Quantification of the time course of retinal degeneration . 10 ommatidia from 5 separate flies of each genotype were scored and plotted . Y - axis is the number of intact rhabdomeres / ommatidium . The maximum value possible is 7 . X - axis is the age of the flies post eclosion . ( G ) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes . Rearing conditions and the age of the flies are indicated on top . ( * CL - Constant Light ) ( H ) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt : : mCherry transgene . Genotypes of the flies are indicated on the left . Rearing conditions and the age of the flies are indicated on top .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative ERG trace from dark reared 0-1 day old flies of the indicated genotype stimulated by 2s flash of green light. The duration of the stimulating light is shown. Y-axis shows the amplitude of the ERG response. X-axis shows the duration of the recording.(B) Graph showing ERG amplitude normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.(C) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH-GFP in the one day old flies of the indicated genotypes (n=10).(D) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.(E) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalised to the intensity of the first image. (n=10 biological replicates). Error bars represent s.e.m.(F) Quantification of the time course of retinal degeneration. 10 ommatidia from 5 separate flies of each genotype were scored and plotted. Y-axis is the number of intact rhabdomeres/ommatidium. The maximum value possible is 7. X-axis is the age of the flies post eclosion.(G) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CL- Constant Light)(H) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt::mCherry transgene. Genotypes of the flies are indicated on the left. Rearing conditions and the age of the flies are indicated on top.


Figure 3 ( A ) ( i ) Representative optical neutralization ( ON ) images showing rhabdomere structure of the indicated genotypes . Rearing conditions and the age of the flies are indicated on top . ( * CD - Constant Dark ) . ( ii ) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9 ; dEsytKO mutants reared in 12 hr L / D cycle ( / ) for 2 days . Scale bar : 1µm ( asterisk indicates degenerated rhabdomere ) . ( B ) Quantification of the time course taken for retinal degeneration . 10 ommatidia were scored from 5 flies of each genotype and plotted . ( C ) Representative ERG traces showing the duration of light pulse , X - axis indicates time in msec and Y - axis indicates the average ERG amplitude in mV . ( D ) Graph showing ERG amplitude of rdgB9 and rdgB9 ; dEsytKO normalized to body weight . Y - axis shows the ratio of ERG amplitude of individual flies to their body weight . X - axis indicates genotypes . Each data point represents an individual fly tested . Error bar represents s . e . m . ( E ) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors . The fluorescence of the PH - GFP probe is depicted . The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light ( a - f ) used for image capture depicted . Representative deep pseudopupil images acquired at specified time points are depicted . Genotypes as indicated . ( F ) Quantification of the mean fluorescence intensity of the PIP2 probe PH - GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes ( n = 10 biological replicates ) . ( G ) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time , X - axis represents the genotypes , Y - axis represent intensity normalized to the intensity of the first image . ( n = 10 biological replicates ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) (i) Representative optical neutralization (ON) images showing rhabdomere structure of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CD- Constant Dark). (ii) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9;dEsytKO mutants reared in 12 hr L/D cycle ( / ) for 2 days. Scale bar: 1µm (asterisk indicates degenerated rhabdomere).(B) Quantification of the time course taken for retinal degeneration. 10 ommatidia were scored from 5 flies of each genotype and plotted.(C) Representative ERG traces showing the duration of light pulse, X-axis indicates time in msec and Y-axis indicates the average ERG amplitude in mV.(D) Graph showing ERG amplitude of rdgB9 and rdgB9;dEsytKO normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.(E) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.(F) Quantification of the mean fluorescence intensity of the PIP2 probe PH-GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes (n=10 biological replicates).(G) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalized to the intensity of the first image. (n=10 biological replicates).


Figure 4 ( A ) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt . Scale bar : 5µm . dEsytKO shows no staining when probed with dEsyt antibody . ( B ) Confocal images showing the localization of exogenously expressed dEsyt : : mCherry protein expressed using the eye specific Rh1 - Gal4 in one day old dark reared flies . Rh1 - Gal4 is shown as a control . A single ommatidium is shown . Scale bar : 5 µm . Phalloidin marks F - actin staining and highlights rhabdomeres R1 - R7 . ( C ) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB . Rearing conditions , age of the flies and genotype is indicated on top of the blot . Tubulin was used as the loading control . ( D , E ) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are ( D ) 1 day old - dark reared and ( E ) 6 days old - exposed to constant illumination . For ( D ) and ( E ) RDGB visualized using an antibody against the endogenous protein . Rhabdomeres are outlined using phalloidin which marks F - actin . Scale bar : 5 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt. Scale bar: 5µm. dEsytKO shows no staining when probed with dEsyt antibody.(B) Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7.(C) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB. Rearing conditions, age of the flies and genotype is indicated on top of the blot. Tubulin was used as the loading control.(D, E) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.


Figure 5 ( A ) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark ( i ) day 1 ( D1 ) and ( iii ) day 14 ( D14 ) , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from the same ommatidium ( ii ) day 1 ( iv ) day 14 . Scale bar : 200 nm . Origin boxes for magnification shown . Arrow in ( Aiv ) indicates the SMC forming an MCS with the microvillar PM . M - Microvillar plasma membrane , S - Sub microvillar cisternae , MCS - Membrane Contact Site , P - Pigment granules . ( B ) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark ( from figure 6A ) , Y - axis indicates the number of MCS / photoreceptor . n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) for ( i ) . n = 9 photoreceptors from 3 separate flies ( R7 ) in ( ii ) . ( C ) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark ( i ) day 1 and ( iii ) day 14 , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from the same ommatidium ( ii ) day1 ( iv ) day 14 . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( D ) Quantification of the number of MCSs per photoreceptor ( from figure 6C ) , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 ) ( E ) TEM images of a single ommatidium from ( i ) wild type and ( iii ) dEsytKO photoreceptors of 0 - 1 day old flies reared in dark , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) ( ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( F ) Quantification of the number of MCS per photoreceptor of wild type day 1 v / s dEsytKO day 1 old flies reared in dark , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark (i) day 1 (D1) and (iii) day 14 (D14), scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day 1 (iv) day 14. Scale bar: 200 nm. Origin boxes for magnification shown. Arrow in (Aiv) indicates the SMC forming an MCS with the microvillar PM. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark (from figure 6A), Y-axis indicates the number of MCS/photoreceptor. n=30 photoreceptors from 3 separate flies (R1-R6) for (i). n=9 photoreceptors from 3 separate flies (R7) in (ii). (C) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark (i) day 1 and (iii) day 14, scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day1 (iv) day 14. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCSs per photoreceptor (from figure 6C), X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7) (E) TEM images of a single ommatidium from (i) wild type and (iii) dEsytKO photoreceptors of 0-1 day old flies reared in dark, scale bar: 1µm (red asterisk indicates R7 photoreceptor) (ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (F) Quantification of the number of MCS per photoreceptor of wild type day 1 v/s dEsytKO day 1 old flies reared in dark, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7).


Figure 6 ( A ) TEM images of a single ommatidium from photoreceptors of ( i ) dEsytKO - day 1 ( iii ) dEsytKO - day 14 flies reared in dark , scale bar : 1 µm except for ( Ai ) : 2 µm ( red asterisk indicates R7 photoreceptor ) . ( A - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Boxes represent the origin boxes for magnification . M - Microvillar plasma membrane , S - Sub microvillar cisternae , MCS - Membrane Contact Site , P - Pigment granules . ( B ) Quantification of the number of MCS per photoreceptor , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor . n = 9 photoreceptors from 3 separate flies for R7 . ( C ) TEM images of a single ommatidium from photoreceptors of ( i ) norpAP24 ; dEsytKO - day 1 ( iii ) norpAP24 ; dEsytKO - day 14 flies reared in dark , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( C - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( D ) Quantification of the number of MCS per photoreceptor , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor . ( i ) n = 30 photoreceptors from 3 separate flies for R1 - R6 . ( ii ) n = 9 photoreceptors from 3 separate flies for R7 . ( E ) TEM images of a single ommatidium from photoreceptors of ( i ) rdgB9 - day1 and ( iii ) rdgB9 ; dEsytKO - day 1 flies reared in dark , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( E - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . ( F ) Quantification of the number of MCS per photoreceptor for R1 - R6 in 1 day old dark reared wild type ( from Figure 5B i ) , rdgB9 and rdgB9 ; dEsytKO flies . n = 30 photoreceptors from 3 separate flies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) TEM images of a single ommatidium from photoreceptors of (i) dEsytKO- day 1 (iii) dEsytKO- day 14 flies reared in dark, scale bar: 1 µm except for (Ai): 2 µm (red asterisk indicates R7 photoreceptor). (A-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Boxes represent the origin boxes for magnification. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. n=9 photoreceptors from 3 separate flies for R7. (C) TEM images of a single ommatidium from photoreceptors of (i) norpAP24;dEsytKO- day 1 (iii) norpAP24;dEsytKO- day 14 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (C-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. (i) n=30 photoreceptors from 3 separate flies for R1-R6. (ii) n=9 photoreceptors from 3 separate flies for R7. (E) TEM images of a single ommatidium from photoreceptors of (i) rdgB9-day1 and (iii) rdgB9;dEsytKO - day 1 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (E-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. (F) Quantification of the number of MCS per photoreceptor for R1-R6 in 1 day old dark reared wild type (from Figure 5B i), rdgB9 and rdgB9;dEsytKO flies. n=30 photoreceptors from 3 separate flies.