Figure 3A . Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR - Cas9 lentiviral vectors with scramble sequence ( sgControl ) or against LDHA ( sgLDHA ) , LDHB ( sgLDHB ) or both ( sgLDHA / B ) . Knockout ( KO ) cells were exposed to 21 % or 1 % O2 for 48 h . D . Left : P3 spheroid growth recorded over one week at 1 % O2 ( n = 3 independent experiments , one experiment including 16 spheroids per condition ) . Data are represented as mean and analyzed using Kruskal - Wallis test followed by Dunn ' s multiple comparisons test . Spheroid growth : sgCont vs sgLDHA , P = 0 . 0003 ; sgCont vs sgLDHB , P = 0 . 78 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Dead area : sgCont vs sgLDHA , P = 0 . 19 ; sgCont vs sgLDHB , P < 0 . 0001 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Right : Viability of spheroids at one week , incubated with calcein ( green ) or ethidium homodimer - 1 ( red ) . Scale bar : 200 µm . E . P3 spheroid invasion in collagen I gel , incubated 24 h at 21 % or 0 . 1 % O2 . Invasion rate is expressed as fold change from controls ( n = 4 independent experiments , one experiment including 6 - 8 spheroids per condition ) . Data are represented as mean and analyzed using Two - way ANOVA following by Tukey ' s multiple comparisons test : sgCont 21 % vs sgLDHA 21 % , P = 0 . 003 ; sgCont 0 . 1 % vs sgLDHA 0 . 1 % , P < 0 . 0001 ; sgCont 21 % vs sgLDHB 21 % , P < 0 . 0001 ; sgCont 0 . 1 % vs sgLDHB 0 . 1 % , P < 0 . 0001 ; sgCont 21 % vs sgLDHA / B 21 % , P < 0 . 0001 ; sgCont 0 . 1 % vs sgLDHA / B 0 . 1 % , P < 0 . 0001 . Images of representative of control or LDHA / B KO spheroids . Scale bar : 100 µm . F - G . A first cohort of mice was sacrificed when one mouse reached a limit point , brains was extract , snap frozen in liquid nitrogen , sliced and stained . For the second cohort , each mouse was sacrificed when it reached a limit point allowing the draw of survival curves . Left : Immunofluorescence staining of Nestin ( top and middle ) and CD31 ( bottom ) of control and LDHA / B KO P3 tumor section . Scale bars : 2 mm ( top ) , 250 µm ( middle ) and 100 µm ( bottom ) . Right : Graphs represent tumor core and invasion area of control and LDHA / B KO P3 tumors in mm2 ( n = 5 mice per group , average of 5 - 6 sections per tumor ) . Data are represented as mean . For F , data are analyzed using One - way ANOVA following by Dunnett ' s multiple comparisons test . Core : sgCont vs sgLDHA , P = 0 . 48 ; sgCont vs sgLDHB , P = 0 . 99 . Invasion : sgCont vs sgLDHA , P = 0 . 40 ; sgCont vs sgLDHB , P = 0 . 81 . For G , data are analyzed using unpaired t test . Core : sgCont vs sgLDHA / B , P = 0 . 005 . Invasion : sgCont vs sgLDHA / B , P = 0 . 01 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O 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 3A. Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR-Cas9 lentiviral vectors with scramble sequence (sgControl) or against LDHA (sgLDHA), LDHB (sgLDHB) or both (sgLDHA/B). Knockout (KO) cells were exposed to 21% or 1% O2 for 48 h.D. Left: P3 spheroid growth recorded over one week at 1% O2 (n = 3 independent experiments, one experiment including 16 spheroids per condition). Data are represented as mean and analyzed using Kruskal-Wallis test followed by Dunn's multiple comparisons test. Spheroid growth: sgCont vs sgLDHA, P=0.0003; sgCont vs sgLDHB, P=0.78; sgCont vs sgLDHA/B, P<0.0001. Dead area: sgCont vs sgLDHA, P=0.19; sgCont vs sgLDHB, P<0.0001; sgCont vs sgLDHA/B, P<0.0001. Right: Viability of spheroids at one week, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 200 µm.E. P3 spheroid invasion in collagen I gel, incubated 24 h at 21% or 0.1% O2. Invasion rate is expressed as fold change from controls (n = 4 independent experiments, one experiment including 6-8 spheroids per condition). Data are represented as mean and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont 21% vs sgLDHA 21%, P=0.003; sgCont 0.1% vs sgLDHA 0.1%, P<0.0001; sgCont 21% vs sgLDHB 21%, P<0.0001; sgCont 0.1% vs sgLDHB 0.1%, P<0.0001; sgCont 21% vs sgLDHA/B 21%, P<0.0001; sgCont 0.1% vs sgLDHA/B 0.1%, P<0.0001. Images of representative of control or LDHA/B KO spheroids. Scale bar: 100 µm.F-G. A first cohort of mice was sacrificed when one mouse reached a limit point, brains was extract, snap frozen in liquid nitrogen, sliced and stained. For the second cohort, each mouse was sacrificed when it reached a limit point allowing the draw of survival curves. Left: Immunofluorescence staining of Nestin (top and middle) and CD31 (bottom) of control and LDHA/B KO P3 tumor section. Scale bars: 2 mm (top), 250 µm (middle) and 100 µm (bottom). Right: Graphs represent tumor core and invasion area of control and LDHA/B KO P3 tumors in mm2 (n = 5 mice per group, average of 5-6 sections per tumor). Data are represented as mean. For F, data are analyzed using One-way ANOVA following by Dunnett's multiple comparisons test. Core: sgCont vs sgLDHA, P=0.48; sgCont vs sgLDHB, P=0.99. Invasion: sgCont vs sgLDHA, P=0.40; sgCont vs sgLDHB, P=0.81. For G, data are analyzed using unpaired t test. Core: sgCont vs sgLDHA/B, P=0.005. Invasion: sgCont vs sgLDHA/B, P=0.01.


Figure 5A . Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21 % or 0 . 1 % O2 . Roman numbers indicate the respiratory complex number . The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21 % and 0 . 1 % O2 ( n = 3 independent experiments ) . B . Epifluorescence ( top ) and quantitative image analysis ( bottom ) of immune stained mitochondria ( TOM20 , green ) from LDH KO P3 cells ( Phalloidin , red ; DAPI , blue ) upon exposure to 0 . 1 % O2 ( n = 2 independent experiments , 28 - 32 cells per group ) . Data are represented as mean and analyzed using One - way ANOVA following by Tukey ' s multiple comparisons test . Mitochondrial mass : sgCont vs sgLDHA / B , P = 0 . 005 ; sgLDHA / sgLDHB vs sgLDHA / B , p = 0 . 006 . Network aspect ratio : sgCont vs sgLDHA / B , P = 0 . 03 ; sgLDHA vs sgLDHA / B , P = 0 . 02 ; sgLDHB vs sgLDHA / B , p = 0 . 04 . Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area . Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network . Scale bar : 10 µm . E . Kaplan - Meier survival curves of xenotransplanted mice with LDHA / B KO ( red ) or control ( blue ) P3 cells , treated with phenformin or irradiated with 10 Gy ( n = 6 - 8 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) test : sgCont Vehicle vs sgCont Phenformin , P = 0 . 019 ; sgCont Vehicle vs sgCont Irradiation , P = 0 . 0002 ; sgCont Irradiation vs sgLDHA / B Irradiation , P = 0 . 024 .

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 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 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

Figure 5A. Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21% or 0.1% O2. Roman numbers indicate the respiratory complex number. The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21% and 0.1% O2 (n = 3 independent experiments).B. Epifluorescence (top) and quantitative image analysis (bottom) of immune stained mitochondria (TOM20, green) from LDH KO P3 cells (Phalloidin, red; DAPI, blue) upon exposure to 0.1% O2 (n = 2 independent experiments, 28-32 cells per group). Data are represented as mean and analyzed using One-way ANOVA following by Tukey's multiple comparisons test. Mitochondrial mass: sgCont vs sgLDHA/B, P=0.005; sgLDHA/sgLDHB vs sgLDHA/B, p=0.006. Network aspect ratio: sgCont vs sgLDHA/B, P=0.03; sgLDHA vs sgLDHA/B, P=0.02; sgLDHB vs sgLDHA/B, p=0.04. Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area. Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network. Scale bar: 10 µm.E. Kaplan-Meier survival curves of xenotransplanted mice with LDHA/B KO (red) or control (blue) P3 cells, treated with phenformin or irradiated with 10 Gy (n = 6-8 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: sgCont Vehicle vs sgCont Phenformin, P=0.019; sgCont Vehicle vs sgCont Irradiation, P=0.0002; sgCont Irradiation vs sgLDHA/B Irradiation, P=0.024.


Figure 6D . Left : P3 spheroid growth recorded over 72 h during incubation with stiripentol at 0 . 1 % O2 ( n = 3 independent experiments , one experiment including 8 spheroids per condition ) . Data are represented as mean ± s . d . and growth at 72 h are analyzed using unpaired t test : P < 0 . 0001 . Right : Viability of spheroids at 72 h , incubated with calcein ( green ) or ethidium homodimer - 1 ( red ) . Scale bar : 100 µm . E . Left : P3 spheroid invasion in collagen I gel , incubated 24 h with 500 µM stiripentol at 0 . 1 % O2 . Invasion rate is expressed as fold change from control ( n = 3 independent experiments , one experiment including 8 spheroids per condition ) . Data are represented as mean and analyzed using unpaired t test : P < 0 . 0001 . Right : Representative images of invasive spheroids for each condition . Scale bar : 100 µm . F . Kaplan - Meier survival curves of xenotransplanted mice with P3 cells . Mice were treated either with vehicle ( blue ) or stiripentol ( red ) ( n = 15 - 16 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) test : P = 0 .

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 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

Figure 6D. Left: P3 spheroid growth recorded over 72 h during incubation with stiripentol at 0.1% O2 (n = 3 independent experiments, one experiment including 8 spheroids per condition). Data are represented as mean ± s.d. and growth at 72 h are analyzed using unpaired t test: P<0.0001. Right: Viability of spheroids at 72 h, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 100 µm. E. Left: P3 spheroid invasion in collagen I gel, incubated 24 h with 500 µM stiripentol at 0.1% O2. Invasion rate is expressed as fold change from control (n = 3 independent experiments, one experiment including 8 spheroids per condition). Data are represented as mean and analyzed using unpaired t test: P<0.0001. Right: Representative images of invasive spheroids for each condition. Scale bar: 100 µm.F. Kaplan-Meier survival curves of xenotransplanted mice with P3 cells. Mice were treated either with vehicle (blue) or stiripentol (red) (n = 15-16 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: P=0.002


Figure 1A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( A ) left untreated or treated with increasing concentrations of LLO for 15 min . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) . ( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( B ) left uninfected ( U ) or infected with wt or Δhly Lm for 1 h . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) . ( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( C ) infected with wt Lm for the indicated time points . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) . ( D ) Confocal microscopy images of HeLa cells : ( D ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) , immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) and stained with DAPI ( blue ) . Arrows point to various NMHCIIA - Gp96 positive cortical bundles in different cell . Insets show high magnification image ( E ) Quantification of Pearson ' s correlation coefficient ( PCC ) within : uninfected cells ( U ) , LLO - treated cells , cortical NMHCIIA bundles or equivalent size control cellular ROI . ( Ctrl ROI ) Data are mean ± SEM ( n = 6 ) ; p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( F ) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) Lm ( blue ) and stained with DAPI ( white ) . Arrows indicate NMHCIIA - Gp96 cortical bundles at cortical sites close to wt Lm . ( D and F ) Scale bar - 10 μm . See also Figs EV1 and EV2 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (A) left untreated or treated with increasing concentrations of LLO for 15 min. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).(A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (B) left uninfected (U) or infected with wt or Δhly Lm for 1 h. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).(A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (C) infected with wt Lm for the indicated time points. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).(D) Confocal microscopy images of HeLa cells: (D) left untreated or treated with LLO (0.5 nM, 15 min), immunolabelled for NMHCIIA (green), Gp96 (red) and stained with DAPI (blue). Arrows point to various NMHCIIA-Gp96 positive cortical bundles in different cell. Insets show high magnification image (E) Quantification of Pearson's correlation coefficient (PCC) within: uninfected cells (U), LLO-treated cells, cortical NMHCIIA bundles or equivalent size control cellular ROI. (Ctrl ROI) Data are mean±SEM (n=6); p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.05, **p<0.01, ***p<0.001(F) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA (green), Gp96 (red) Lm (blue) and stained with DAPI (white). Arrows indicate NMHCIIA-Gp96 cortical bundles at cortical sites close to wt Lm. (D and F) Scale bar - 10 μm. See also Figs EV1 and EV2.


Figure 2 ( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( A ) GFP - NMHCIIA . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( A ) Arrows indicate NMHCIIA bundles at PM blebbing sites . ( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( B ) simultaneously GFP - NMHCIIA and mcherry - KDEL . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( B ) Highlights depicting ER structures within NMHCIIA bundles and PM blebs . Arrows indicate cortical ER surrounding NMHCIIA accumulations , arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs ( C ) Confocal microscopy images of HeLa cells left untreated ( Control ) or treated with LLO ( 0 . 5 nM 15 min ) . Cells were stained with FITC - WGA ( Plasma membrane , PM - red ) and immunolabelled for NMHCIIA ( green ) . Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated ( 1 ) or detached ( 2 ) from the cell body . ( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( D ) LLO pre - incubated with cholesterol ( LLOCHT ) ( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( E ) LLO in medium supplemented with 140 mM K + ( HighK + ) and LLO in Ca2 + - free medium Ca2 + ( Ca2 + free ) ( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( F ) LLO in the presence of 25 μM blebbistatin ( BB ) . ( G ) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER - KDEL ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) . ( H ) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA ( green ) and Gp96 ( red ) . Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA . ( I ) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) , SLO ( 1 . 5 μg / ml , 30 min ) ( SLO ) or aerolysin ( 0 . 2 nM , 40 min ) ( AL ) .

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 O O O O O O O O O O O O O O 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 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 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 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

Figure 2(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (A) GFP-NMHCIIA . LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (A) Arrows indicate NMHCIIA bundles at PM blebbing sites.(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (B) simultaneously GFP-NMHCIIA and mcherry-KDEL. LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (B) Highlights depicting ER structures within NMHCIIA bundles and PM blebs. Arrows indicate cortical ER surrounding NMHCIIA accumulations, arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs(C) Confocal microscopy images of HeLa cells left untreated (Control) or treated with LLO (0.5 nM 15 min). Cells were stained with FITC-WGA (Plasma membrane, PM-red) and immunolabelled for NMHCIIA (green). Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated (1) or detached (2) from the cell body.(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT)(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (E) LLO in medium supplemented with 140 mM K+(HighK+) and LLO in Ca2+-free medium Ca2+ (Ca2+free)(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (F) LLO in the presence of 25 μM blebbistatin (BB).(G) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER-KDEL (blue), NMHCIIA (green) and Gp96 (red).(H) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA (green) and Gp96 (red). Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA.(I) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL).


Figure 3 ( A ) Confocal microscopy images of shCtrl , shGp96 or shNMHCIIA HeLa cells treated with LLO ( 0 . 5 nM , 15 min ) and immunolabelled for KDEL - proteins ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) . Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells . Scale bar - 10 μm . ( B - D ) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with ( B ) LLO ( 0 . 5 nM , 15 min ) , ( C ) aerolysin ( 0 . 2 nM , 40 min ) or ( D ) with wt Lm . Values are the mean ± SEM ( n ≥ 3 ) , p - values were calculated using ( B - C ) one - way - ANOVA with Dunnett post hoc analyses and ( D ) two - tailed un - paired Student ' s t - test , * p < 0 . 5 , * * * p < 0 . 001 . For shNMHCIIA , bundles were detected following actin staining ( Fig EV3C ) . ( E - F ) Number of ( E ) blebs per cell or ( F ) retracting blebs per cell evaluated by time - lapse microscopy analysis of LLO - treated shCtrl or shGp96 cells . shCtrl n = 32 cells and shGp96 n = 40 cells , p - values were calculated using two - tailed un - paired Student ' s t - test * p < 0 . 5 . ( G ) Sequential frames of time - lapse microscopy analysis of LLO - treated HeLa cells expressing GFP - NMHCIIA ( shCtrl , shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl - BB ) . LLO was added to culture medium 10 seconds before t0 . ( H ) Immunoblots of Ser19 - phosphorylated MRLC ( pMRLC ) , MRLC and actin levels from shCtrl or shGp96 cells left untreated ( U ) or treated with 0 . 1 nM LLO for the indicated time points . Quantification of pMRLC levels corresponds to the mean ± SEM ( n ≥ 3 ) , p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 , * * * p < 0 . 001 . See also Appendix Fig S2 and Fig EV3 .

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 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 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

Figure 3(A) Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with LLO (0.5 nM, 15 min) and immunolabelled for KDEL-proteins (blue), NMHCIIA (green) and Gp96 (red). Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells. Scale bar - 10 μm. (B-D) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with (B) LLO (0.5 nM, 15 min), (C) aerolysin (0.2 nM, 40 min) or (D) with wt Lm. Values are the mean ±SEM (n≥3), p-values were calculated using (B-C) one-way-ANOVA with Dunnett post hoc analyses and (D) two-tailed un-paired Student's t-test, *p<0.5, ***p<0.001. For shNMHCIIA, bundles were detected following actin staining (Fig EV3C).(E-F) Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n=32 cells and shGp96 n=40 cells, p-values were calculated using two-tailed un-paired Student's t-test *p<0.5. (G) Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFP-NMHCIIA (shCtrl, shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl-BB). LLO was added to culture medium 10 seconds before t0.(H) Immunoblots of Ser19-phosphorylated MRLC (pMRLC), MRLC and actin levels from shCtrl or shGp96 cells left untreated (U) or treated with 0.1 nM LLO for the indicated time points. Quantification of pMRLC levels corresponds to the mean ± SEM (n≥3), p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01, ***p<0.001. See also Appendix Fig S2 and Fig EV3.


Figure 4 ( Ai ) Confocal microscopy images of LLO - treated HeLa cells immunolabelled for the indicated proteins . Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles . ( Aii ) Quantification of Pearson ' s correlation coefficient ( PCC ) between NMHCIIA and the indicated proteins within entire LLO - treated cells , cortical bundles or equivalent size control cellular ROI ( Ctrl ROI ) . For Calpain 2 cortical bundles were defined by actin staining . Data are representative of one experiment repeated 3 independent times with similar results . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 ; * * * p < 0 . 001 , ns - non significant ( B ) Immunoblots of NMHCIIA , Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) ( LLO ) . ( C ) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with 25 μg / ml of Wnt5a for the indicated time . ( D ) Confocal microscopy images of HeLa cells untreated ( Mock ) or treated with 25 μg / ml of Wnt5a for 30 min . Cells were immunolabelled for ER - KDEL ( blue ) , Gp96 ( red ) and NMHCIIA ( green ) . Arrows indicate polarised localization of NMHCIIA , ER - KDEL and Gp96 in Wnt5a - treated cells . ( E - F ) Confocal microscopy images of shCtlr , shGp96 or shNMHCIIA HeLa cells stained for actin ( red ) and DAPI ( blue ) and immunolabelled for ( E ) NMHCIIA ( green ) or ( F ) focal adhesion kinase ( FAK ) ( green ) . ( A , D - F ) Scale bar - 10 μm . Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points . Quantification of the % of cells with ( G ) stress fibers or ( H ) focal adhesion points labelled by FAK . ( I - J ) Wound closure assay . ( I ) Sequential frames of time - lapse microscopy of HeLa cells grown to confluence separated by a stopper . Stopper was removed at t0 and cellular migration was imaged for the indicated times . ( J ) Quantification of the % of wound area occupied by migrating cells over time stopper removal ( upper panel ) and respective rate of closure ( lower panel ) .

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 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 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 O O O O O O O O O O O O O O O O O O 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

Figure 4(Ai) Confocal microscopy images of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles. (Aii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent size control cellular ROI (Ctrl ROI). For Calpain 2 cortical bundles were defined by actin staining. Data are representative of one experiment repeated 3 independent times with similar results. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses ** p<0.01; *** p<0.001, ns - non significant(B) Immunoblots of NMHCIIA, Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO).(C) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time.(D) Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarised localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells.(E-F) Confocal microscopy images of shCtlr, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). (A, D-F) Scale bar - 10 μm. Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points. Quantification of the % of cells with (G) stress fibers or (H) focal adhesion points labelled by FAK.(I-J) Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the % of wound area occupied by migrating cells over time stopper removal (upper panel) and respective rate of closure (lower panel).


Figure 5 ( A ) Epifluorescence microscopy images of supernatants from LLO - treated ( 0 . 5 nM 15 min ) shCtlr , shGp96 or shNMHCIIA HeLa cells collected into poly - lysine - coated cover slips , fixed and stained with FITC - WGA ( green ) and DAPI ( blue ) . Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei . Scale bar - 10 μm . ( B ) Quantification of released PM blebs ( total FITCWGA / field ) . Values are the means ± SEM ( n ≥ 3 ) . * * * p < 0 . 001 . ( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( C ) 0 . 5 nM LLO for the indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . ( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( D ) 1 . 5 or 3 nM LLO for 15 min ; or ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . ( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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

Figure 5(A) Epifluorescence microscopy images of supernatants from LLO-treated (0.5 nM 15 min) shCtlr, shGp96 or shNMHCIIA HeLa cells collected into poly-lysine-coated cover slips, fixed and stained with FITC-WGA (green) and DAPI (blue). Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei. Scale bar - 10 μm. (B) Quantification of released PM blebs (total FITCWGA/field). Values are the means ± SEM (n≥3). ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (C) 0.5 nM LLO for the indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (D) 1.5 or 3 nM LLO for 15 min; or (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


Figure 6 ( A - B ) Epifluorecence microscopy images of HeLa cells ( A ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) . Cells were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) and stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable bacteria . ( A - B ) Epifluorecence microscopy images of HeLa cells ( B ) infected with GFP - expressing wt Lm for 6 h . Cells were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) and stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable bacteria . ( C ) Quantification of the % of infected cells with exofacial - PS sites in HeLa cells infected with wt Lm for 6 h . Data are the mean ± SEM ( n = 3 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 . ( D - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa and RAW264 . 7 cells , infected with GFP - expressing wt or Δhly Lm for 6 h . The levels of PI incorporation by uninfected cells were subtracted from infected samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria ( Fig Appendix Fig S3 ) .

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 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 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 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

Figure 6(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.(A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.(C) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5.(D-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa and RAW264.7 cells, infected with GFP-expressing wt or Δhly Lm for 6 h. The levels of PI incorporation by uninfected cells were subtracted from infected samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria (Fig Appendix Fig S3).


Figure 7 ( A ) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates ( WCL ) and IP fractions ( IP Gp96 ) of extracts of zebrafish larvae 3 dpf uninfected ( U ) or infected with wt or ∆ hly Lm ( low dose 200 - 1500 CFU ) for 24h . ( B ) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control ( Ctrl - mo ) or Gp96 morpholino oligonucleotides ( Gp96 mo ) . ( C ) Confocal microscopy images of zebrafish larvae ( Ctrl mo or Gp96 mo ) infected ( low dose ) in the tail muscle , with the GFP - expressing wt or Δhly Lm for 24 h , immunolabelled for KDEL - proteins ( green ) , and stained with phalloidin ( actin , red ) and DAPI ( white ) . Images show actin rich structures at the cell cortex ( arrows ) and ER - KDEL puncta ( arrowheads ) in larvae infected with wt strain . ( D ) CFU counts per zebrafish larvae ( Ctrl or Gp96 mo ) infected with wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm and analysed at 0 , 24 , 48 hpi . Results are mean ± SEM ( n ≥ 3 ) ( horizontal bars ) and each circle represents 1 larvae . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 . ( E ) Survival curves of zebrafish larvae ( Ctrl - mo or Gp96 - mo ) infected with a wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm . Results are the mean ± SEM ( n ≥ 3 ) . wt infection of Ctrl and Gp96 mo , n = 28 larvae ; Δhly infection of Gp96 mo n = 13 . p - values were calculated using Log Rank test , * * p < 0 . 01 . ( F ) CFU counts per zebrafish larvae infected with a high dose of ∆ hly Lm at different times post - infection . Results are mean ± SEM ( n ≥ 3 ) ( horizontal bars ) ; each point represents 1 larvae . See also Fig EV5 .

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 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 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 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 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

Figure 7(A) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates (WCL) and IP fractions (IP Gp96) of extracts of zebrafish larvae 3 dpf uninfected (U) or infected with wt or ∆hly Lm (low dose 200-1500 CFU) for 24h.(B) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control (Ctrl-mo) or Gp96 morpholino oligonucleotides (Gp96 mo).(C) Confocal microscopy images of zebrafish larvae (Ctrl mo or Gp96 mo) infected (low dose) in the tail muscle, with the GFP-expressing wt or Δhly Lm for 24 h, immunolabelled for KDEL-proteins (green), and stained with phalloidin (actin, red) and DAPI (white). Images show actin rich structures at the cell cortex (arrows) and ER-KDEL puncta (arrowheads) in larvae infected with wt strain.(D) CFU counts per zebrafish larvae (Ctrl or Gp96 mo) infected with wt (low dose) or Δhly (high dose > 10,000 CFU) Lm and analysed at 0, 24, 48 hpi. Results are mean ± SEM (n≥3) (horizontal bars) and each circle represents 1 larvae. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01.(E) Survival curves of zebrafish larvae (Ctrl-mo or Gp96-mo) infected with a wt (low dose) or Δhly (high dose > 10,000 CFU) Lm. Results are the mean ± SEM (n≥3). wt infection of Ctrl and Gp96 mo, n=28 larvae; Δhly infection of Gp96 mo n=13. p-values were calculated using Log Rank test, **p<0.01.(F) CFU counts per zebrafish larvae infected with a high dose of ∆hly Lm at different times post-infection. Results are mean ± SEM (n≥3) (horizontal bars); each point represents 1 larvae. See also Fig EV5.


Figure 1 ( F ) Examples of expression levels of Treg signature genes Foxp3 , Il10ra , Nrp1 , Ikzf4 and Ccr6 ; and examples of expression levels of genes upregulated in Tet2 / 3 DKO Treg cells Eomes , Ccl4 , Chek1 and Fn1 . The Y - axis shows FPKM ( Fragment Per Kilobase per Million reads ) , the X - axis shows the different cell types . Error bars show mean ± S . D . from two ( CD4 + naïve T cells ) or four ( WT and Tet2 / 3 DKO Treg ) biological replicates .

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Figure 1(F) Examples of expression levels of Treg signature genes Foxp3, Il10ra, Nrp1, Ikzf4 and Ccr6; and examples of expression levels of genes upregulated in Tet2/3 DKO Treg cells Eomes, Ccl4, Chek1 and Fn1. The Y-axis shows FPKM (Fragment Per Kilobase per Million reads), the X-axis shows the different cell types. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT and Tet2/3 DKO Treg) biological replicates.


Figure 2 ( D ) Genome browser view of CMS - IP data showing CMS ( 5hmC ) distribution at Il2ra and Foxp3 locus for CD4 + naïve T cells and WT Treg cells . Hydroxymethylated regions ( hmRs ) and DhmRs are shown on the top ; conservation ( cons ) is shown at the bottom .

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Figure 2(D) Genome browser view of CMS-IP data showing CMS (5hmC) distribution at Il2ra and Foxp3 locus for CD4+ naïve T cells and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom.


Figure 3 ( E ) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4 + naïve T cells , WT Treg cells and Tet2 / 3 DKO Treg cells . Differentially hydroxymethylated regions ( DhmRs ) and differentially methylated CpGs ( DM - CpGs ) are shown on top of the tracks . Conservation track ( Cons ) and denotation of all CpGs are shown at the bottom . WGBS data are from two biological replicate samples .

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Figure 3(E) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Differentially hydroxymethylated regions (DhmRs) and differentially methylated CpGs (DM-CpGs) are shown on top of the tracks. Conservation track (Cons) and denotation of all CpGs are shown at the bottom. WGBS data are from two biological replicate samples.


Figure 4 ( E ) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4 + naïve T cells , WT Treg cells and Tet2 / 3 DKO Treg cells . DhmRs , DM - CpGs and DARs ( shaded ) are shown on top of the tracks . Conservation track ( Cons ) is shown at the bottom .

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Figure 4(E) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. DhmRs, DM-CpGs and DARs (shaded) are shown on top of the tracks. Conservation track (Cons) is shown at the bottom.


Figure 5 ( H - J ) Box - and - whisker plots of DNA methylation levels for DM - CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs ( H ) , DM - CpGs less methylated in WT Treg cells vs CD4 + naïve T cells ( I ) and DM - CpGs less methylated in WT Treg cells vs Tet2 / 3 DKO Treg cells ( J ) . DM - CpGs denotes the CpGs in the respective cell types used for the comparison . Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range . P - values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey ' s post - hoc test . Data are from two biological replicates .

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Figure 5(H-J) Box-and-whisker plots of DNA methylation levels for DM-CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated in WT Treg cells vs CD4+ naïve T cells (I) and DM-CpGs less methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.


Figure 7 ( D ) Graphs depicting the percentage of viable cells for TGFβ iTregs ( left ) and TGFβ + RA + VitC iTregs ( right ) differentiated with 0 U / ml , 1 U / ml , 5 U / ml and 10 U / ml IL - 2 . Error bars show mean ± S . D . from three biological replicates .

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Figure 7(D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates.


Figure 8 ( C ) ChIP - qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U / ml IL - 2 restimulation . GMPR is a negative control region . Data are from two biological replicates .

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Figure 8(C) ChIP-qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U/ml IL-2 restimulation. GMPR is a negative control region. Data are from two biological replicates.


Figure 1 ( D , E ) Imaging of luciferase activity in the liver of HIF1aAlbKO ( D ) and HIF2aAlbKO ( E ) mice after a hydrodynamic tail vein injection with a HRE - luc reporter plasmid or control ( PBS ) according to body weight followed by 6h normoxia or hypoxia . ( F , G ) Bioluminescent photon counts normalized to the PBS control group in liver of ( F ) HIF1aAlbKO and ( G ) HIF2aALbKO mice and their wild - type littermates 6h after normoxia of hypoxia . Data information : All bars represent mean ± SEM . Each individual data point represent individual mice . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 . N = normoxia , H = hypoxia . Signal around the perimeter of the Petri dishes represent aspecific luciferase signal .

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Figure 1(D, E) Imaging of luciferase activity in the liver of HIF1aAlbKO (D) and HIF2aAlbKO (E) mice after a hydrodynamic tail vein injection with a HRE-luc reporter plasmid or control (PBS) according to body weight followed by 6h normoxia or hypoxia. (F, G) Bioluminescent photon counts normalized to the PBS control group in liver of (F) HIF1aAlbKO and (G) HIF2aALbKO mice and their wild-type littermates 6h after normoxia of hypoxia. Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N = normoxia, H = hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal.


Figure 2 ( E ) Scatter plot showing log fold change ( LFC ) of all DEX - upregulated genes ( LFC > 1 and P ≤ 0 . 05 ) and DEX - downregulated genes ( LFC < - 1 and P ≤ 0 . 05 ) in normoxia vs hypoxia after 24h . The black line represents the diagonal , and the red line represents the real slope ± standard error of the data as analysed by linear regression . ( F , G ) Examples of GR - responsive genes based on the RNA - seq data 6h ( F ) and 24h ( G ) after normoxia or hypoxia and DEX stimulation . Data information : All bars represent mean ± SEM . N = 3 per group . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 . ( H ChIP - seq on liver derived from mice which were subjected to hypoxia ( 6h and 24h ) , followed by DEX ( 10 mg / kg ) injection and 2h later sacrificed for liver isolation . ( H ) Box plots showing the LFC of genes responsive to DEX in both normoxia or hypoxia ( 6h and 24h ) . The central band represents the median . The box ranges from the first quartile ( Q1 ) to the third quartile ( Q3 ) which represents the interquartile range ( IQR = Q3 - Q1 ) and covers the central 50 % of the data . The whiskers illustrate the minimum ( Q1 - 1 . 5 * IQR ) and maximum ( Q3 + 1 . 5 * IQR ) of the data . Outliers are shown as dots . N = 3 biological replicates per condition .

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Figure 2(E) Scatter plot showing log fold change (LFC) of all DEX-upregulated genes (LFC > 1 and P ≤ 0.05) and DEX-downregulated genes (LFC < -1 and P ≤ 0.05) in normoxia vs hypoxia after 24h. The black line represents the diagonal, and the red line represents the real slope ± standard error of the data as analysed by linear regression.(F, G) Examples of GR-responsive genes based on the RNA-seq data 6h (F) and 24h (G) after normoxia or hypoxia and DEX stimulation. Data information: All bars represent mean ± SEM. N=3 per group. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01.(H ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (H) Box plots showing the LFC of genes responsive to DEX in both normoxia or hypoxia (6h and 24h). The central band represents the median. The box ranges from the first quartile (Q1) to the third quartile (Q3) which represents the interquartile range (IQR = Q3 - Q1) and covers the central 50% of the data. The whiskers illustrate the minimum (Q1 - 1.5*IQR) and maximum (Q3 + 1.5*IQR) of the data. Outliers are shown as dots. N=3 biological replicates per condition.


Figure 3Female C57BL / 6J mice were put in normoxia or hypoxia for 6h or 24h , injected i . p . with PBS or DEX ( 10 mg / kg ) and livers were isolated for genome - wide transcriptomics via RNA - seq . N = 3 per group for a single RNA - seq . ( A , B ) Examples of DEX - responsive genes in hypoxia 6h ( A ) and 24h ( B ) which were not induced by DEX in normoxia . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 . ChIP - seq on liver derived from mice which were subjected to hypoxia ( 6h and 24h ) , followed by DEX ( 10 mg / kg ) injection and 2h later sacrificed for liver isolation . ( E ) Examples of specific GR DNA - binding peaks , associated with the DEX - induced gene Slc22a5 in hypoxia ( 6h and 24h ) . ( F ) HIF1aHIF2afl / fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h and stimulated with DEX . Slc25a30 expression was measured in the liver via RT - qPCR . N = 3 per group , one experiment . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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Figure 3Female C57BL/6J mice were put in normoxia or hypoxia for 6h or 24h, injected i.p. with PBS or DEX (10 mg/kg) and livers were isolated for genome-wide transcriptomics via RNA-seq. N=3 per group for a single RNA-seq. (A, B) Examples of DEX-responsive genes in hypoxia 6h (A) and 24h (B) which were not induced by DEX in normoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (E) Examples of specific GR DNA-binding peaks, associated with the DEX-induced gene Slc22a5 in hypoxia (6h and 24h).(F) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h and stimulated with DEX. Slc25a30 expression was measured in the liver via RT-qPCR. N=3 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.


Figure 4 ( C ) Heatmap representing log2 values of shared stress GRE genes ( counts ) induced by hypoxia 6h and 24h and induced by DEX in normoxia ( LFC > 1 and P ≤ 0 . 05 ) . ( D - G ) Female C57BL / 6J and ADX mice were put in normoxia or hypoxia for 6h or 24h , n = 6 / group , two independent experiments . ( D , E ) Confirmation of RNA - seq data via RT - qPCR [ 6h ( D ) or 24h ( E ) ] . ( F , G ) Plasma GC concentration [ 6h ( F ) or 24h ( G ) ] . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 . ( M ) HIF1aHIF2afl / fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h . Stress GRE genes were measured in the liver via RT - qPCR . N = 4 per group , one experiment . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 . ( N ) Expression levels of DEX responsive genes in normoxia and unique DEX responsive genes induced by hypoxia were measured in the liver of ADX mice after DEX stimulation during hypoxia ( 24h ) via RT - qPCR . N = 3 - 4 per group , one experiment . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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Figure 4(C) Heatmap representing log2 values of shared stress GRE genes (counts) induced by hypoxia 6h and 24h and induced by DEX in normoxia (LFC > 1 and P ≤ 0.05).(D-G) Female C57BL/6J and ADX mice were put in normoxia or hypoxia for 6h or 24h, n=6/group, two independent experiments. (D, E) Confirmation of RNA-seq data via RT-qPCR [6h (D) or 24h (E)]. (F, G) Plasma GC concentration [6h (F) or 24h (G)]. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.(M) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h. Stress GRE genes were measured in the liver via RT-qPCR. N=4 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.(N) Expression levels of DEX responsive genes in normoxia and unique DEX responsive genes induced by hypoxia were measured in the liver of ADX mice after DEX stimulation during hypoxia (24h) via RT-qPCR. N=3-4 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


Figure 5 ( D ) HIF1α and HIF2α protein levels were analysed via western blot using VINCULIN as a loading control . HIF1α and HIF2α protein levels were quantified using FIJI and normalized to VINCULIN levels . P - values were calculated using Mann - Whitney test . N = 5 biological replicates per group . N = normoxia , H = hypoxia . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

Figure 5(D) HIF1α and HIF2α protein levels were analysed via western blot using VINCULIN as a loading control. HIF1α and HIF2α protein levels were quantified using FIJI and normalized to VINCULIN levels. P-values were calculated using Mann-Whitney test. N=5 biological replicates per group. N = normoxia, H = hypoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, **P<0.01, *P≤0.05.


Figure 6 ( A ) Female C57BL / 6J and ADX mice were put in normoxia and hypoxia for 6h and 24h . Plasma was isolated for metabolomics analysis . The heatmap represents log10 of metabolites which are significantly increased of the plasma of C57BL / 6J mice ( C57BL / 6J P ≤ 0 . 05 and ADX P > 0 . 05 , LFC > 1 ) , significantly increased in the plasma of C57BL / 6J and ADX mice ( C57BL / 6J P ≤ 0 . 05 and ADX P ≤ 0 . 05 , LFC > 1 ) , and significantly increased in the plasma of ADX mice only ( C57BL / 6J P > 0 . 05 and ADX P ≤ 0 . 05 , LFC > 1 ) . ( D - G ) FFA levels and stress GRE genes were determined in the plasma and iWAT of female C57BL / 6J mice injected with 5 mg RU486 or vehicle ( DMSO ) ( D , F ) , and in GRdim / dim mice and their wild - type littermates ( E , G ) after 6h of normoxia and hypoxia . N = 4 - 9 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 . ( H - J ) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL / 6J and ADX mice ( H ) , in female C57BL / 6J mice injected with 5 mg RU486 or vehicle ( DMSO ) ( I ) , and in GRdim / dim mice and their wild - type littermates ( J ) . N = 5 - 9 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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 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 O O O O O O O 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) Female C57BL/6J and ADX mice were put in normoxia and hypoxia for 6h and 24h. Plasma was isolated for metabolomics analysis. The heatmap represents log10 of metabolites which are significantly increased of the plasma of C57BL/6J mice (C57BL/6J P ≤ 0.05 and ADX P > 0.05, LFC > 1), significantly increased in the plasma of C57BL/6J and ADX mice (C57BL/6J P ≤ 0.05 and ADX P ≤ 0.05, LFC > 1), and significantly increased in the plasma of ADX mice only (C57BL/6J P > 0.05 and ADX P ≤ 0.05, LFC > 1).(D-G) FFA levels and stress GRE genes were determined in the plasma and iWAT of female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (D, F), and in GRdim/dim mice and their wild-type littermates (E, G) after 6h of normoxia and hypoxia. N=4-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.(H-J) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL/6J and ADX mice (H), in female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (I), and in GRdim/dim mice and their wild-type littermates (J). N=5-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


Figure 7 ( A ) Female C57BL / 6J mice ( n = 3 per group ) were put in normoxia or hypoxia for 24h , injected with PBS or DEX ( 10 mg / kg ) and 2h later , indicated organs were isolated and gene expression was measured via RT - qPCR . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . Survival curves were analysed with Fisher ' s exact test . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 . ( B ) LPS LD100 dose - response in female C57BL / 6J mice injected with indicated LPS doses after 24h hypoxia . During the follow - up of lethality , mice remained under hypoxic conditions . N - values are indicated in the legend . ( E ) Female C57BL / 6J mice were injected with 14 . 5 mg / kg LPS ( normoxia ) or 5 mg / kg LPS ( hypoxia ) after 24h normoxia or hypoxia . Liver was isolated 6h after injected and typical GRE genes were measured via RT - qPCR . N = 4 - 5 per group . ( F ) Female ADX mice were put in normoxia or hypoxia for 24h , injected i . p . with PBS or DEX ( 10 mg / kg ) and 2h later , liver was isolated for RT - qPCR analyses of typical GRE genes . N = 3 - 4 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . Survival curves were analysed with Fisher ' s exact test . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 . ( G ) Female C57BL / 6J mice were injected with 14 . 5 mg / kg LPS ( normoxia ) or 5 mg / kg LPS ( hypoxia ) after 24h normoxia or hypoxia , with or without pre - treatment with 10 mg / kg DEX 1h before LPS injection . During the follow - up of lethality , mice remained under normoxic or hypoxic conditions . Mice in normoxia : black circles ( LPS ) , black squares ( DEX - LPS ) ; mice in hypoxia : white circles ( LPS ) , white squares ( DEX - LPS ) . ( H ) Female ADX mice were injected with 0 . 05 mg / kg LPS after 24h normoxia or hypoxia , , with or without 10 mg / kg DEX pre - treatment 1h before LPS injection . During the follow - up of lethality , mice remained under normoxic or hypoxic conditions . ADX mice in normoxia : black circles ( LPS ) , black squares ( DEX - LPS ) ; ADX mice in hypoxia : white circles ( LPS ) , white squares ( DEX - LPS ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7(A) Female C57BL/6J mice (n=3 per group) were put in normoxia or hypoxia for 24h, injected with PBS or DEX (10 mg/kg) and 2h later, indicated organs were isolated and gene expression was measured via RT-qPCR. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.(B) LPS LD100 dose-response in female C57BL/6J mice injected with indicated LPS doses after 24h hypoxia. During the follow-up of lethality, mice remained under hypoxic conditions. N-values are indicated in the legend.(E) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia or hypoxia. Liver was isolated 6h after injected and typical GRE genes were measured via RT-qPCR. N=4-5 per group. (F) Female ADX mice were put in normoxia or hypoxia for 24h, injected i.p. with PBS or DEX (10 mg/kg) and 2h later, liver was isolated for RT-qPCR analyses of typical GRE genes. N=3-4 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.(G) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia or hypoxia, with or without pre-treatment with 10 mg/kg DEX 1h before LPS injection. During the follow-up of lethality, mice remained under normoxic or hypoxic conditions. Mice in normoxia: black circles (LPS), black squares (DEX-LPS); mice in hypoxia: white circles (LPS), white squares (DEX-LPS).(H) Female ADX mice were injected with 0.05 mg/kg LPS after 24h normoxia or hypoxia, , with or without 10 mg/kg DEX pre-treatment 1h before LPS injection. During the follow-up of lethality, mice remained under normoxic or hypoxic conditions. ADX mice in normoxia: black circles (LPS), black squares (DEX-LPS); ADX mice in hypoxia: white circles (LPS), white squares (DEX-LPS).


Figure 1 ( B ) The Western blot analysis of GFP , Spd2 and αTub ( loading control ) in the brain extracts from wild type ( Oregon R , OR ) , Spd2WT - OE and Spd2DK - OE third instar larva . Representative immune blot images are shown on the left . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control of each biological replicate ( n = 2 ) and their means are shown as dots and bars , respectively , in a scatted dot plot on the right . The numbers above the bars indicate means ± Range . GFP - Spd2DK proteins in Spd2DK - OE larval brain extracts showed approximately 2 . 04 - fold higher accumulation than GFP - Spd2WT proteins in Spd2WT - OE brain extracts . ( C ) The Western blot analysis of Spd2 , CycB and αTub in OR , fzrRNAi , Spd2WT - RES and Spd2DK - RES brain extracts . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control and their means are presented in a scatted dot plot on the right ( biological replicate n = 2 ) . Means ± Range are shown above the bars . RFP - Spd2DK proteins in Spd2DK - RES larval brain extracts showed approximately 2 . 08 - fold higher accumulation than RFP - Spd2WT proteins in Spd2WT - RES brain extracts . ( D ) Larval brains of OR , Spd2WT - RES , fzrRNAi and Spd2WT - OE flies were stained by an anti - Spd2 antibody . Representative images of NBs are shown on the top . Scale bar : 10 µm . The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured , and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots ( see Materials and Methods for detail ) . The lines indicate means ± SD . The numbers of NBs ( n ) and the numbers of brains analysed ( N ) are control : n = 29 ( N = 6 ) , Spd2WT - RES : n = 30 ( N = 4 ) , fzrRNA : n = 30 ( N = 6 ) , and Spd2WT - OE : n = 27 ( N = 10 ) . P values were calculated using non - parametric two - tailed Mann - Whitney tests . Based on these results , the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype ( or control ) : Spd2WT - RES : fzrRNAi : Spd2DK - RES : Spd2WT - OE : Spd2DK - OE = 1 : 1 . 00 ~ 1 . 18 : 1 . 06 ~ 1 . 29 : 2 . 08 ~ 2 . 45 ( = 1 . 00 ~ 1 . 18 x 2 . 08 ) : 1 . 93 ~ 3 . 62 : 3 . 94 ~ 7 . 38 ( = 1 . 93 ~ 3 . 62 x 2 .

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 O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B) The Western blot analysis of GFP, Spd2 and αTub (loading control) in the brain extracts from wild type (Oregon R, OR), Spd2WT-OE and Spd2DK-OE third instar larva. Representative immune blot images are shown on the left. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control of each biological replicate (n = 2) and their means are shown as dots and bars, respectively, in a scatted dot plot on the right. The numbers above the bars indicate means ± Range. GFP-Spd2DK proteins in Spd2DK-OE larval brain extracts showed approximately 2.04-fold higher accumulation than GFP-Spd2WT proteins in Spd2WT-OE brain extracts. (C) The Western blot analysis of Spd2, CycB and αTub in OR, fzrRNAi, Spd2WT-RES and Spd2DK-RES brain extracts. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control and their means are presented in a scatted dot plot on the right (biological replicate n = 2). Means ± Range are shown above the bars. RFP-Spd2DK proteins in Spd2DK-RES larval brain extracts showed approximately 2.08-fold higher accumulation than RFP-Spd2WT proteins in Spd2WT-RES brain extracts.(D) Larval brains of OR, Spd2WT-RES, fzrRNAi and Spd2WT-OE flies were stained by an anti-Spd2 antibody. Representative images of NBs are shown on the top. Scale bar: 10 µm. The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured, and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots (see Materials and Methods for detail). The lines indicate means ± SD. The numbers of NBs (n) and the numbers of brains analysed (N) are control: n= 29 (N = 6), Spd2WT-RES: n = 30 (N = 4), fzrRNA: n = 30 (N = 6), and Spd2WT-OE: n = 27 (N = 10). P values were calculated using non-parametric two-tailed Mann-Whitney tests. Based on these results, the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype (or control) : Spd2WT-RES: fzrRNAi: Spd2DK-RES: Spd2WT-OE: Spd2DK-OE = 1: 1.00~1.18: 1.06~1.29: 2.08~2.45 (= 1.00~1.18 x 2.08): 1.93~3.62: 3.94 ~ 7.38 (= 1.93~3.62 x 2.04


Figure 2 ( C ) In time lapse movies , NBs that showed an active centrosome that was attached to the cortex ( " Attached " ) and NBs with both two centrosomes being inactivated and detached from the cortex ( " Detached " ) in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs . The numbers of NBs analysed from at least four brains ( n ) are Control : 29 , fzrRNAi : 80 , Spd2WT - RES : 45 , Spd2DK - RES : 52 , Spd2WT - OE : 43 , Spd2DK - OE : 40 , Spd2WT - OE + fzrRNAi : 72 . p - values are fzrRNAi : 0 . 0069 , Spd2WT - RES : 0 . 2479 , Spd2DK - RES : 0 . 0116 , Spd2WT - OE : < 0 . 0001 , Spd2DK - OE : < 0 . 0001 , Spd2WT - OE + fzrRNAi : 0 . 0008 ( Pearson ' s chi - squared tests were performed between control and each condition ) . ( E ) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT - RES and Spd2DK - RES larval brains , and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes ( see Materials and Method ) in a scatted dot plot . Red bars represent means ± SD . The numbers of NBs analysed from at least 3 different brains of Spd2WT - RES and Spd2DK - RES ( n ) were 40 and 40 for Asl , 40 and 41 for RFP - Spd2 , 42 and 37 for Cnn , 40 and 41 for γTub , and 40 and 40 for AurA .

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 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

Figure 2(C) In time lapse movies, NBs that showed an active centrosome that was attached to the cortex ("Attached") and NBs with both two centrosomes being inactivated and detached from the cortex ("Detached") in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs. The numbers of NBs analysed from at least four brains (n) are Control: 29, fzrRNAi: 80, Spd2WT-RES: 45, Spd2DK-RES: 52, Spd2WT-OE: 43, Spd2DK-OE: 40, Spd2WT-OE + fzrRNAi: 72. p-values are fzrRNAi: 0.0069, Spd2WT-RES: 0.2479, Spd2DK-RES: 0.0116, Spd2WT-OE: <0.0001, Spd2DK-OE: <0.0001, Spd2WT-OE + fzrRNAi: 0.0008 (Pearson's chi-squared tests were performed between control and each condition).(E) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT-RES and Spd2DK-RES larval brains, and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes (see Materials and Method) in a scatted dot plot. Red bars represent means ± SD. The numbers of NBs analysed from at least 3 different brains of Spd2WT-RES and Spd2DK-RES (n) were 40 and 40 for Asl, 40 and 41 for RFP-Spd2, 42 and 37 for Cnn, 40 and 41 for γTub, and 40 and 40 for AurA.


Figure 3 ( D ) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ( " attached " ) and those with centrosomes being detached from the cortex ( " detached " ) for each line , using the same dataset as in Fig . 3C . The same control data as in Fig . 3C is shown for reference . fzrRNA detached ( mean ± SD : 34 . 62 ° ± 23 . 39 ) , Spd2DK - OE detached ( 64 . 01 ° ± 33 . 93 ) , and Spd2WT - OE + fzrRNAi attached ( 39 . 55 ° ± 27 . 08 ) and detached ( 59 . 08 ° ± 34 . 88 ) brains showed statistically significant differences from control , while NBs with cortically attached centrosomes in any of the lines did not show significant differences . n : the number of NB divisions analysed from at least four brains for each line . p - values were calculated using Mann - Whitney U tests comparing with control and are shown above the dots .

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

Figure 3(D) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ("attached") and those with centrosomes being detached from the cortex ("detached") for each line, using the same dataset as in Fig. 3C. The same control data as in Fig. 3C is shown for reference. fzrRNA detached (mean ± SD: 34.62° ± 23.39), Spd2DK-OE detached (64.01° ± 33.93), and Spd2WT-OE+fzrRNAi attached (39.55° ± 27.08) and detached (59.08° ± 34.88) brains showed statistically significant differences from control, while NBs with cortically attached centrosomes in any of the lines did not show significant differences. n: the number of NB divisions analysed from at least four brains for each line. p-values were calculated using Mann-Whitney U tests comparing with control and are shown above the dots.


Figure 4 ( B ) Selected images from time - lapse microscopy of NBs in control ( wor > mCh - Tub , pUbq - GFP - Fzr , top panels ) and Spd2DK - OE ( lower panels ) larval brains ( Movies EV1 , EV10 ) . In the control NB , the daughter centrosomes ( blue arrowheads ) first matured ( - 5 . 0 min ) and was segregated into the daughter NB ( 12 . 0 min ) , while the mother centrosome ( yellow arrowheads ) matured a little later ( 0 . 0 min ) and segregated into the GMC . In contrast , in the Spd2DK - OE NB , both centrosomes lost microtubule nucleation activity in interphase ( red arrowheads ) . The centrosome that matured earlier ( blue arrowheads , - 6 . 0 min ) was incorrectly segregated into the GMC ( 12 . 0 min ) while the centrosome that matured later ( yellow arrowheads , 0 . 0 min ) was segregated into the NB ( 12 . 0 min ) . Dotted white lines outline the dividing NBs and the newly formed GMC , which are also marked by asterisks .

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

Figure 4(B) Selected images from time-lapse microscopy of NBs in control (wor>mCh-Tub, pUbq-GFP-Fzr, top panels) and Spd2DK-OE (lower panels) larval brains (Movies EV1, EV10). In the control NB, the daughter centrosomes (blue arrowheads) first matured (-5.0 min) and was segregated into the daughter NB (12.0 min), while the mother centrosome (yellow arrowheads) matured a little later (0.0 min) and segregated into the GMC. In contrast, in the Spd2DK-OE NB, both centrosomes lost microtubule nucleation activity in interphase (red arrowheads). The centrosome that matured earlier (blue arrowheads, -6.0 min) was incorrectly segregated into the GMC (12.0 min) while the centrosome that matured later (yellow arrowheads, 0.0 min) was segregated into the NB (12.0 min). Dotted white lines outline the dividing NBs and the newly formed GMC, which are also marked by asterisks.


Figure 5 ( C , D ) Representative images of interphase NBs in fixed wor > lacZ ( control , top panels ) and HA - Spd2WT - OE larval brains expressing Polo - GFP from the native promoter and stained for DAPI and Spd2 ( C ) . Asymmetric indexes of Polo - GFP in interphase NBs wor > lacZ ( control , top panels ) and HA - Spd2WT - OE larval brains ( D ) . 38 and 39 NBs ( n ) from at least three brains were analysed in each line . The asymmetric distribution of Polo - GFP at interphase centrosomes was unaffected by HA - Spd2WT overexpression . ( E ) Representative images of interphase NBs in fixed mCh - Spd2WT - RES ( top panels ) and mCH - Spd2DK - RES larval brains and stained for DAPI , γTub and Cnn . Both multichannel and single - channel images of mCh - Spd2 , γTub and Cnn are shown . Dotted yellow squares highlight centrosomes and their magnified images are shown in insets . Similar to Spd2DK - RES NBs , in mCh - Spd2CONS - RES NBs , Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh - Spd2WT - RES NBs , but Cnn was still asymmetrically distributed . ( G , H ) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT - RES and Spd2DK - OE NBs . ( G ) Representative images from time - lapse movies ( Movies EV13 , EV14 ) of Spd2 fluorescent signals in Spd2WT - RES NBs ( top panels ) and Spd2DK - OE NBs ( lower panels ) upon photobleaching . ( H ) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT - RES ( n = 7 ) and Spd2DK - OE NBs ( n = 5 ) . Means ± SEM of the normalised fluorescent intensities are shown in a line graph . Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK - OE NBs than in Spd2WT - RES NBs .

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 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 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

Figure 5(C, D) Representative images of interphase NBs in fixed wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains expressing Polo-GFP from the native promoter and stained for DAPI and Spd2 (C). Asymmetric indexes of Polo-GFP in interphase NBs wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains (D). 38 and 39 NBs (n) from at least three brains were analysed in each line. The asymmetric distribution of Polo-GFP at interphase centrosomes was unaffected by HA-Spd2WT overexpression.(E) Representative images of interphase NBs in fixed mCh-Spd2WT-RES (top panels) and mCH-Spd2DK-RES larval brains and stained for DAPI, γTub and Cnn. Both multichannel and single-channel images of mCh-Spd2, γTub and Cnn are shown. Dotted yellow squares highlight centrosomes and their magnified images are shown in insets. Similar to Spd2DK-RES NBs, in mCh-Spd2CONS-RES NBs, Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh-Spd2WT-RES NBs, but Cnn was still asymmetrically distributed.(G, H) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT-RES and Spd2DK-OE NBs. (G) Representative images from time-lapse movies (Movies EV13, EV14) of Spd2 fluorescent signals in Spd2WT-RES NBs (top panels) and Spd2DK-OE NBs (lower panels) upon photobleaching. (H) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT-RES (n = 7) and Spd2DK-OE NBs (n = 5). Means ± SEM of the normalised fluorescent intensities are shown in a line graph. Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK-OE NBs than in Spd2WT-RES NBs.


Figure 1H . Endogenous levels of Stim1 in testes from C57BL / 6 wt and splice deficient 10A mice , using an antibody detecting the N - term of STIM1 , detecting STIM1A ( A , red arrow ) , STIM1 and its glycosylated ( wt ) and unglycosylated ( wt gl . ) form .

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Figure 1H. Endogenous levels of Stim1 in testes from C57BL/6 wt and splice deficient 10A mice, using an antibody detecting the N-term of STIM1, detecting STIM1A (A, red arrow), STIM1 and its glycosylated (wt) and unglycosylated (wt gl.) form.