Figure 6 ( A ) Representative confocal imaging of localization of Lamp1 - mcherry and LysoTracker Green in wild type ( w1118 ) , NimB4 sk2 and draper △ 5 macrophages ( live imaging ) . The arrows indicate the colocalization ( w1118 ) or the clustering ( NimB4sk2 and draper △ 5 ) of Lamp1 - mcherry and LysoTracker Green signals . Scale bar : 10 μm . ( B ) Quantification of the colocalization of Lamp1 - mcherry with LysoTracker Green , as measured by Pearson ' s correlation coefficient between the two signals . Values from at least five independent experiments are represented as mean ± SD ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) . ( C ) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo ™ Red . Wild type ( w1118 ) , NimB4sk2 and draper △ 5 macrophages from L3 wandering larvae were incubated with pHrodo ™ Red apoptotic cells for 60 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using pHrodoTM Red S . aureus BioparticlesTM conjugates . Wild type , NimB4sk2 and NimC11 , eater1 Hml ∆ - Gal4 > UAS - GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S . aureus BioparticlesTM for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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Figure 6(A) Representative confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). (C) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 7 ( A - B ) Representative fluorescence microscopy images of wild type ( A ) or NimB4sk2 ( B ) macrophages expressing Rab5 ( middle panel ) or Rab7 ( right panel ) driven by Hml ∆ - Gal4 and stained with the LysoTracker Red ( live imaging ) . Overlay of fluorescence and DIC . Scale bar = 10 μm . ( C ) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae ( confocal live imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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Figure 7(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 1Same as ( E ) with amiRhen1 - 14 scions and non - transgenic WT or hen1 - 14 rootstocks . Average relative U6 - normalized band - intensity quantifications from two biological replicates are indicated .

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Figure 1Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.


Figure 2Phenotype of pSUC2 : : amiRSUL plants in hst - 1 / - 25 / - 26 backgrounds . RT - qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes . Error - bars : SD . Welch ' s t - test p - values are indicated . n = 5 .

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Figure 2Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds.RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.


Figure 3amiRSUL northern analysis in input and AGO1 - immunoprecipitated ( AGO1 - IP ) fractions isolated from leaves of pSUC2 : : amiRSUL plants in the specified genotypes . No Ab : No antibody added to the HST input extract ( IP negative control ) . miR165 / 166 and U6 were probed as endogenous controls . AGO1 western analysis and Coomassie blue ( Coom . ) staining of the western blot membrane are provided as controls . amiRSUL band - intensity quantifications , U6 - normalized for input samples , are indicated .

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Figure 3amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.


Figure 4amiRSUL northern analysis , in biological duplicates , in scions ( S ) and rootstocks ( R ) in the specified genotypes ' grafting combinations ( amiR : pSUC2 : : amiRSUL ) ( see also Appendix Fig . S12B ) . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated for each tissue . amiRSUL northern analysis , in biological duplicates , from total RNA extracted from aphids fed on WT or amiR plants , in the specified genetic backgrounds . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated .

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Figure 4amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue.amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.


Figure 5Localization of HST : GFP and , as a reference , of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves ( C ) or roots ( D ) . CW : calcofluor white staining . Scale bars : 20 µm . Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2 : : HST : GFP , pSUC2 : : GFP or pSUC2 : : tmGFP9 . Scale bars : 100 µm . Confocal fluorescence images of the differentiation and division zones of pSUC2 : : GFPhst - 1 or pSUC2 : : tmGFP9hst - 1 root tips . Scale bars : 100 µm .

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Figure 5Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm. Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm.


Figure 6Subcellular localizations of HST : GFP expressed from the UBQ10 promoter ( pUBQ ) in Arabidopsis roots ( i ) . Distinct planes of the same root cells imaged by confocal microscopy are shown ( ii - iii ) . PI : propidium iodide staining . Scale bars : 50µm ( i ) ; 10µm ( ii - iii ) . Subcellular localizations of hst - 3 : GFP expressed from pUBQ in root cells . Distinct planes of the same root cells imaged by confocal microscopy are shown ( i - ii ) . PI : as in ( A ) . Scale bars : 10µm .

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Figure 6Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.


Figure 7miR395 northern analysis in scions ( S ) and hyl1 - 2 rootstocks ( R ) , in the indicated genotypes ' combinations , under SO4 - sufficient ( + SO4 ) or SO4 - starved ( - SO4 ) conditions , in two biological replicates ( rep . ) . U6 was probed as an endogenous control . Relative U6 - normalized band - intensity quantifications are indicated for each tissue in - SO4 conditions . miR160 northern analysis in Meselect - separated leaf vasculature and epidermis in WT versus hst - 1 plants , in three biological replicates ( rep . ) . U6 : as in ( A ) . Relative U6 - normalized band - intensity quantifications are indicated . Basic fuchsin staining of protoxylem ( unfilled arrowheads ) and metaxylem ( filled arrowheads ) in WT , shr - 2 or hst - 1 roots . * : protoxylem gap . Scale bars : 10 µm .

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Figure 7miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated.Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.


Figure 1B . Survival frequencies observed after DSB induction at TEL6R in WT , dnl4Δ , sir4Δ cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . C . Survival frequencies observed after DSB induction at LYS2 in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) . F . Representative images of Mre11 - YFP foci in response to an I - SceI - induced DSB at LYS2 in WT cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Scale bars are 2 μm . G . Quantification of cells with DSB induced Mre11 - YFP foci after DSB induction at LYS2 I - SceI cleavage site in WT , sae2Δ and Sir3 overexpressing ( oeSir3 ) strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) . H . Survival frequencies after DSB induction at LYS2 locus , in strains where SIR3 is expressed from its native , pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid . Fold increase in Sir3 protein by pAHD1 or pGPD is indicated . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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Figure 1B. Survival frequencies observed after DSB induction at TEL6R in WT, dnl4Δ, sir4Δ cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Error bars indicate survival standard error (SEM) of at least three independent experiments. C. Survival frequencies observed after DSB induction at LYS2 in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).F. Representative images of Mre11-YFP foci in response to an I-SceI-induced DSB at LYS2 in WT cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Scale bars are 2 μm. G. Quantification of cells with DSB induced Mre11-YFP foci after DSB induction at LYS2 I-SceI cleavage site in WT, sae2Δ and Sir3 overexpressing (oeSir3) strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).H. Survival frequencies after DSB induction at LYS2 locus, in strains where SIR3 is expressed from its native, pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid. Fold increase in Sir3 protein by pAHD1 or pGPD is indicated. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


Figure 2A . Representative images of Sir3 - mCherry and Sae2 - GFP signal in WT and SIR3 overexpressing cells . Scale bars are 2 μm . B . Sir3 - binding at TEL6R in untagged , WT , sir3Δ cells or in cells overexpressing Sir3 ( oeSir3 ) . Binding is probed by ChIP - qPCR 0 . 2 ( red arrows ) and 1kb ( blue arrows ) from telomeres and at the OGG1 control locus using antibodies against Sae2 - GFP . The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates ( SEM ) . C . Co - immunoprecipitation between Sir3 and Sae2 - GFP from cells overexpressing Sir3 , analysed by Western blot with anti - GFP and anti - Sir3 antibodies . D . Co - immunoprecipitation between Sir3 and Sae2 - GFP from WT cells using antibodies against Sae2 - GFP , analysed by Western blot .

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Figure 2A. Representative images of Sir3-mCherry and Sae2-GFP signal in WT and SIR3 overexpressing cells. Scale bars are 2 μm.B. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).C. Co-immunoprecipitation between Sir3 and Sae2-GFP from cells overexpressing Sir3, analysed by Western blot with anti-GFP and anti-Sir3 antibodies. D. Co-immunoprecipitation between Sir3 and Sae2-GFP from WT cells using antibodies against Sae2-GFP, analysed by Western blot.


Figure 3D . Yeast two - hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells . Growth on - His + 3AT and blue coloration on X - gal indicate an interaction .

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Figure 3D. Yeast two-hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT and blue coloration on X-gal indicate an interaction.


Figure 4B . Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C . C . Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C .

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Figure 4B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C. C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.


Figure 5A . Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) . B . Representative images of Sae2 - GFP in WT cells and in cells overexpressing either full - length Sir3 or the sir3SaID domain . Scale bars are 2 μm . F . Representative silver - stained gels of in vitro GST - pulldown of GST or GST - Sir3SaID , GST - sir3 - T557ISaID and Sae2C purified peptides . Control : Sae2C ( 300 ng , lane 6 ) . G . Co - immunoprecipitation between Sae2 - GFP and Sir3 from untagged , Sae2 - GFP WT cells , and Sae2 - GFP cells overexpressing WT Sir3 ( oeSir3 , WT ) , Sae2 - GFP sir3Δ or Sae2 - GFP overexpressing the sir3 - T557I mutant ( oeSir3 , T557I ) using antibodies against Sae2 - GFP , analysed by Western blot with anti - GFP and anti - Sir3 antibodies .

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Figure 5A. Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).B. Representative images of Sae2-GFP in WT cells and in cells overexpressing either full-length Sir3 or the sir3SaID domain. Scale bars are 2 μm.F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).G. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP WT cells, and Sae2-GFP cells overexpressing WT Sir3 (oeSir3, WT), Sae2-GFP sir3Δ or Sae2-GFP overexpressing the sir3-T557I mutant (oeSir3, T557I) using antibodies against Sae2-GFP, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.


Figure 6A . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) . C . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression . Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) . E . Representative images of two hybrid assays testing the interaction between the full - length Sir3 and full - length Sae2 proteins in WT cells expressing or not high levels of Sir4 ( oeSir4 and WT respectively ) .

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Figure 6A. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).C. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression. Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).E. Representative images of two hybrid assays testing the interaction between the full-length Sir3 and full-length Sae2 proteins in WT cells expressing or not high levels of Sir4 (oeSir4 and WT respectively).


Figure 7A . Relative fold enrichment of Sae2 at 0 . 2 kb from I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . B . Relative fold enrichment of Sae2 at 0 . 2 kb from TEL6R after DSB induction at the LYS2 I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) . D . Survival frequencies after DSB induction at LYS2 locus in sae2 - E267E mutant cells overexpressing or not full - length Sir3 ( oeSir3 ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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Figure 7A. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).D. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


Figure 1Schematic , western blot , and immunofluorescence analysis of the 3 × FLAG - UBXD8 knockin U2OS cell line . Scale bar , 5 μm . Data information : In C , mitochondria were visualized by anti - Tom20 immunostaining ; ER was labeled by anti - PDI immunostaining .

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Figure 1Schematic, western blot, and immunofluorescence analysis of the 3×FLAG-UBXD8 knockin U2OS cell line. Scale bar, 5 μm. Data information: In C, mitochondria were visualized by anti-Tom20 immunostaining; ER was labeled by anti-PDI immunostaining.


Figure 2Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆ UBXD8 HeLa cells . Immunoprecipitation analysis of UBXD8 interaction with mitochondrial ( MARCH5 and MUL1 ) and ER ( RNF185 ) ubiquitin E3 ligases . HeLa cells were infected with lentivirus to stably express HA - tagged ubiquitin E3 ligases . Whole cell lysates were prepared for anti - HA immunoprecipitation . Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185 . Whole cell lysates from 3 × FLAG - UBXD8 knockin HEK293T cells were prepared for anti - FLAG immunoprecipitation . MARCH5 and RNF185 : short exposure for whole cell lysate blots , long exposure for FLAG - IP blots . Fractionation analysis of UBXD8 ' s effect on VCP recruitment to mitochondria and the ER . Whole cell lysate , mitochondria , and ER fractions were purified from WT , ∆ UBXD8 , and ∆ UBXD8 HeLa cells rescued with UBXD8 or the UBX * mutant . UBX * - UBXD8 carries four point mutations ( K367A , F407A , P408G , R409A ) in the UBX domain to disrupt the UBXD8 - VCP interaction . For each sample , 5 μg proteins were loaded for western blot analysis . Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells . Data are shown as mean ± SE from three biological repeats . Statistics : one - way ANOVA ; * * P < 0 . 01 , * * * P < 0 . 001 .

O B-PANEL_START 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 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 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

Figure 2Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆UBXD8 HeLa cells.Immunoprecipitation analysis of UBXD8 interaction with mitochondrial (MARCH5 and MUL1) and ER (RNF185) ubiquitin E3 ligases. HeLa cells were infected with lentivirus to stably express HA-tagged ubiquitin E3 ligases. Whole cell lysates were prepared for anti-HA immunoprecipitation.Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185. Whole cell lysates from 3×FLAG-UBXD8 knockin HEK293T cells were prepared for anti-FLAG immunoprecipitation. MARCH5 and RNF185: short exposure for whole cell lysate blots, long exposure for FLAG-IP blots.Fractionation analysis of UBXD8's effect on VCP recruitment to mitochondria and the ER. Whole cell lysate, mitochondria, and ER fractions were purified from WT, ∆UBXD8, and ∆UBXD8 HeLa cells rescued with UBXD8 or the UBX* mutant. UBX*-UBXD8 carries four point mutations (K367A, F407A, P408G, R409A) in the UBX domain to disrupt the UBXD8-VCP interaction. For each sample, 5 μg proteins were loaded for western blot analysis.Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells. Data are shown as mean ± SE from three biological repeats. Statistics: one-way ANOVA; **P < 0.01, ***P < 0.001.


Figure 3A , B . Western blot ( A ) and quantitative ( B ) analyses of MiD49 and Mcl1 degradation in WT and ∆ MARCH5 HeLa cells . Cycloheximide ( CHX : 200 μg / ml ; protein synthesis inhibitor ) ; MG132 : 20 μM , proteasome inhibitor . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . C , D . Western blot ( C ) and quantitative ( D ) analyses of Mcl1 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . E , F . Western blot ( E ) and quantitative ( F ) analyses of MiD49 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats F ) . Statistics : two - tailed unpaired Student ' s t - test F ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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Figure 3A, B. Western blot (A) and quantitative (B) analyses of MiD49 and Mcl1 degradation in WT and ∆MARCH5 HeLa cells. Cycloheximide (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome inhibitor. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.C, D. Western blot (C) and quantitative (D) analyses of Mcl1 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.E, F. Western blot (E) and quantitative (F) analyses of MiD49 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats F). Statistics: two-tailed unpaired Student's t-test F); *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


Figure 4Fractionation analysis of the subcellular localization of mito - UBXD8 and ER - UBXD8 . Whole cell lysate , mitochondria , and ER fractions were purified from the indicated HeLa cells . Exogenously - expressed mito - UBXD8 has similar expression level with endogenous UBXD8 in WT cells ; exogenously - expressed WT UBXD8 and ER - UBXD8 have similar expression levels . D , E . Western blot ( D ) and quantitative ( E ) analyses of Mcl1 degradation in the indicated HeLa cells . ∆ UBXD8 HeLa cells were rescued with vector , WT - , mito - , and ER - UBXD8 . Data are shown as mean ± SE from three biological repeats ( E ) . Statistics : two - tailed unpaired Student ' s t - test ( E ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 4Fractionation analysis of the subcellular localization of mito-UBXD8 and ER-UBXD8. Whole cell lysate, mitochondria, and ER fractions were purified from the indicated HeLa cells. Exogenously-expressed mito-UBXD8 has similar expression level with endogenous UBXD8 in WT cells; exogenously-expressed WT UBXD8 and ER-UBXD8 have similar expression levels.D, E. Western blot (D) and quantitative (E) analyses of Mcl1 degradation in the indicated HeLa cells. ∆UBXD8 HeLa cells were rescued with vector, WT-, mito-, and ER-UBXD8. Data are shown as mean ± SE from three biological repeats (E). Statistics: two-tailed unpaired Student's t-test (E); *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 5Cell survival analysis of the indicated HeLa cells treated with Actinomycin D ( ActD , 1 μM ) or Doxorubicin ( Doxo , 10 μM ) . Representative images are shown . Cell viability was measured by Cell Titer - Glo . Scale bar , 50 μm . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Western blot analysis of apoptosis activation ( caspase cleavage and PARP cleavage ) in the indicated HeLa cells treated similarly as in ( D ) . Cell survival analysis of the indicated HeLa cells . 7 × 105 cells in one well of 6 - well plate were treated with ActD ( 200 nM ) for 4 hours or Doxo ( 50 nM ) for 2 hours and subsequently cultured in normal medium for 21 days . Surviving colonies were stained by 0 . 1 % ( W / V ) methylene blue . Representative images and quantifications of the surviving colony numbers are shown . Data information : Data are shown as mean ± SE from three biological repeats G ) . Statistics : one - way ANOVA ( G ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 5Cell survival analysis of the indicated HeLa cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001.Western blot analysis of apoptosis activation (caspase cleavage and PARP cleavage) in the indicated HeLa cells treated similarly as in (D).Cell survival analysis of the indicated HeLa cells. 7 × 105 cells in one well of 6-well plate were treated with ActD (200 nM) for 4 hours or Doxo (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown. Data information: Data are shown as mean ± SE from three biological repeats G). Statistics: one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 6Western blot analysis of BH3 - domain containing proteins in the indicated HeLa cells treated with Doxo ( 10 μM ) . Three proteins with enhanced level in ∆ UBXD8 cells ( Noxa , Bik , and Bnip3 ) are highlighted in red . Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 . Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats C , Statistics : two - tailed unpaired Student ' s t - test C , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 . Western blot ( D , analysis of the indicated HeLa cells . Western blot F ) analysis of the indicated HeLa cells .

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Figure 6Western blot analysis of BH3-domain containing proteins in the indicated HeLa cells treated with Doxo (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.001, ***P < 0.001.Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats C, Statistics: two-tailed unpaired Student's t-test C, ; *P < 0.05, **P < 0.001, ***P < 0.001.Western blot (D, analysis of the indicated HeLa cells.Western blot F) analysis of the indicated HeLa cells.


Figure 7Representative images of the mitophagy events in the indicated HeLa cells . mtKeima emission excited by 405 nm laser is shown as green ; mtKeima emission excited by 561 nm laser is shown as red . Mitolysosomes are pointed by white arrows . Scale bar , 5 μm . C - F . Quantitative analysis of mitophagy events in the indicated HeLa cells . Only cells with mitophagy were selected to quantify mitolysosome numbers ( D , F ) . Data information : Data are shown as mean ± SE from three biological repeats ( C - F , Statistics : two - tailed unpaired Student ' s t - test ( C - F , ; * * P < 0 . 01 , * * * P < 0 . 001 . I - K , Representative images ( I ) and quantitative analysis of mitophagy events ( J , K ) in the indicated HeLa cells . White arrows point to mitolysosomes . Scale bar , 5 μm . Data information : Data are shown as mean ± SE from three biological repeats J , K , Statistics : two - tailed unpaired Student ' s t - test J , K ) ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 7Representative images of the mitophagy events in the indicated HeLa cells. mtKeima emission excited by 405 nm laser is shown as green; mtKeima emission excited by 561 nm laser is shown as red. Mitolysosomes are pointed by white arrows. Scale bar, 5 μm.C-F. Quantitative analysis of mitophagy events in the indicated HeLa cells. Only cells with mitophagy were selected to quantify mitolysosome numbers (D, F). Data information: Data are shown as mean ± SE from three biological repeats (C-F, Statistics: two-tailed unpaired Student's t-test (C-F, ; **P < 0.01, ***P < 0.001.I-K, Representative images (I) and quantitative analysis of mitophagy events (J, K) in the indicated HeLa cells. White arrows point to mitolysosomes. Scale bar, 5 μm. Data information: Data are shown as mean ± SE from three biological repeats J, K, Statistics: two-tailed unpaired Student's t-test J, K); **P < 0.01, ***P < 0.001.


Figure 6A . Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation . The gray scale indicates the density of dots in a given area of the chart . The dotted contours represent the 0 . 5 , 0 . 75 and 0 . 95 probability contours of the 240 WT replicates . The Pearson correlation is ρ = 0 . 65 , with a 95 % CI = [ 0 . 63 , 0 . 66 ] . The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time . Red dots highlight strainsC . Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain . The cumulative distributions of the fraction of cells with two nucleoids ( blue ) and of the fraction of cells with a constriction degree above 0 . 15 ( red ) were plotted against cell age . Cell age was calculated according to the rank of each cell based on their cell length with the formula $ \ text { age } _ { i } \ left ( F \ right ) = \ - \ frac { ln ( 1 - \ frac { F } { 2 } ) } { ln ( 2 ) } $ , where F represents the fraction of cells with a cell length equal or below the length of cell i ( Wold et al , 1994 ) . D . Same plots as in C for three strains clustering in island 8 with the ΔmatP strain . The WT curves shown in C were plotted in gray for comparison . E . Same plots as in C for two island - 8 strains , ΔycjV and ΔhslU , and the ΔhslV strain , which does not partition in island 8 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation. The gray scale indicates the density of dots in a given area of the chart. The dotted contours represent the 0.5, 0.75 and 0.95 probability contours of the 240 WT replicates. The Pearson correlation is ρ = 0.65, with a 95% CI = [0.63, 0.66]. The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time. Red dots highlight strainsC. Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain. The cumulative distributions of the fraction of cells with two nucleoids (blue) and of the fraction of cells with a constriction degree above 0.15 (red) were plotted against cell age. Cell age was calculated according to the rank of each cell based on their cell length with the formula $\text{age}_{i}\left( F \right) = \ - \frac{ln(1 - \frac{F}{2})}{ln(2)}$, where F represents the fraction of cells with a cell length equal or below the length of cell i (Wold et al, 1994). D. Same plots as in C for three strains clustering in island 8 with the ΔmatP strain. The WT curves shown in C were plotted in gray for comparison. E. Same plots as in C for two island-8 strains, ΔycjV and ΔhslU, and the ΔhslV strain, which does not partition in island 8.


Figure 7B . The cumulative distribution of the proportion of constricting cells for the ftsA * mutant and its parent were plotted against cell age . We measured the degree of constriction of all cells using Oufti . For each strain , we had two independently acquired image sets ( n = 496 and 1 , 168 cells for the WT replicates and n = 692 and 1 , 506 cells for the ftsA * replicates ) . The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p - value of 0 . 01 using a Kruskal - Wallis multi - comparison test . Moreover , Bonferroni - corrected post - hoc pairwise tests did not allow a distinction between WT and ftsA * samples . C . Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only . The contour lines represent the 0 . 5 , 075 and 0 . 95 probability envelopes of the 240 independent WT replicates . The gray color levels indicate the density of points in the vicinity of each strain . The ∆ mraZ strain discussed in the text is highlighted in red .

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

Figure 7B. The cumulative distribution of the proportion of constricting cells for the ftsA* mutant and its parent were plotted against cell age. We measured the degree of constriction of all cells using Oufti. For each strain, we had two independently acquired image sets (n = 496 and 1,168 cells for the WT replicates and n = 692 and 1,506 cells for the ftsA* replicates). The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p-value of 0.01 using a Kruskal-Wallis multi-comparison test. Moreover, Bonferroni-corrected post-hoc pairwise tests did not allow a distinction between WT and ftsA* samples.C. Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only. The contour lines represent the 0.5, 075 and 0.95 probability envelopes of the 240 independent WT replicates. The gray color levels indicate the density of points in the vicinity of each strain. The ∆mraZ strain discussed in the text is highlighted in red.


Figure 1 ( A ) Western blotting of AXL and OPCML protein levels in AXL - expressing , OPCML - null SKOV3 and PEA1 cell lines transduced with OPCML " O " or control Empty vector " E " ( B ) Mammalian 2 - Hybrid assay between OPCML and AXL Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t tests ( C ) Western blotting of the OPCML - GST pull down . Input : 1 / 20 of SKOV3 - Empty whole cell lysate ( D ) Western blotting of the anti - AXL immunoprecipitation . Input : 1 / 100 of SKOV3 - OPCML whole cell lysate . IP : Immunoprecipitated protein , IB : Immunoblotted protein ( E ) Immunostaining of OPCML ( red ) and AXL ( green ) in SKOV3 - OPCML and PEA1 - OPCML cells . Scale bar = 10μm Data i M , ( E ) was performed once due to the limited numbers of primary cells ; ( G ) FRET efficiency for AXL - OPCML interaction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " , using AXL - AXL FRET as a positive control . Cells were labeled with Anti - Axl rabbit ( conjugated to donor probe ) and Anti - OPCML mouse ( conjugated to acceptor probe ) , or Anti - Axl mouse ( conjugated to acceptor probe ) and Anti - Axl rabbit ( conjugated to donor probe ) Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t tests ( H ) PLA of AXL - OPCML ( red ) in SKOV3 and PEA1 cells transduced with empty vector " E " or OPCML " O " . Scale bar = 50μmPrimary ovarian tumour cells were characterised for AXL and OPCML levels by ( I ) Western blottin PAX8 was used to identify ovarian cancer cellsPrimary ovarian tumour cells were characterised for AXL and OPCML levels b ( J ) Immunostaining . PAX8 was used to identify ovarian cancer cells ( K ) Quantitation of AXL - OPCML PLA in primary cells Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t tests ( L ) Western blotting of membrane fractionation of SKOV3 - Empty cells : total cell lysate " T " , liquid - disordered soluble fraction " S " , detergent resistant membrane fraction " R " . Caveolin - 1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction , E : SKOV3 - Empty , O : SKOV3 - OPCML ( M ) Densitometry of AXL band intensities in ( J ) in S and R , normalized to AXL intensity in input . Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

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

Figure 1 (A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E" (B) Mammalian 2-Hybrid assay between OPCML and AXL Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests(C) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate(D) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein(E) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm Data i M, (E) was performed once due to the limited numbers of primary cells;(G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe) Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests(H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μmPrimary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blottin PAX8 was used to identify ovarian cancer cellsPrimary ovarian tumour cells were characterised for AXL and OPCML levels b (J) Immunostaining. PAX8 was used to identify ovarian cancer cells(K) Quantitation of AXL-OPCML PLA in primary cells Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests(L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML(M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input. Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


Figure 2 SKOV3 - OPCML cells stimulated with Gas6 over a 12 hour time course and ( A ) stained with DAPI ( cyan ) , Anti - OPCML ( green ) and Anti - AXL ( red ) antibodies ( scale bar = 10μm ) ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n = 3 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t testsd ; ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n = 3 ( E ) Quantitation OPCML - AXL PLA interaction in primary ovarian tumour cells , n = 1 Data i M , ( E ) was performed once due to the limited numbers of primary cells ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( F ) Co - immunoprecipitation with anti - AXL antibody in cells stimulated with Gas6 as indicated . Input : 1 / 50 of whole cell lysate from SKOV3 - OPCML cells . IP : Immunoprecipitated protein , IB : Immunoblotted protein ( G ) Co - immunoprecipitation with anti - pAXL antibody in SKOV3 - OPCML cells stimulated with Gas6 ( H ) OPCML - GST pull down assay in SKOV3 - Empty cells stimulated with Gas6 ( I ) Membrane fractionation in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6 stimulation ( K ) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( L ) Diffusion Co - efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( M ) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( N ) FRET Efficiency in SKOV3 - OPCML " O " cells stimulated with Gas6 , and labeled with Anti - Axl ( donor probe ) and Anti - OPCML ( acceptor probe ) Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( O ) Western blotting of the " S " and " R " membrane fractions in SKOV3 - Empty " E " and OCPML " O " cells stimulated with Gas6 for 3 hours , for Gas6 , AXL and pAXL . Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ±

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

Figure 2 SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm) (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t testsd; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3(E) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1 Data i M, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein(G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6(H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6(I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation(K) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(L) Diffusion Co-efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe) Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(O) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM


Figure 5 ( A ) Filipin staining of SKOV3 - OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0 , 30 minutes , and 12 hours in serum free ( SF ) medium ( B ) Western blotting of SKOV3 - OPCML cells treated with MBCD and separated into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6 stimulation ( C ) Ratio of AXL band intensity in " R " or pAXL band intensity in " S " relative to total AXL " T " from SKOV3 - OPCML " O " in ( B ) ( D ) MBCD treatment of SKOV3 - EMPTY and SKOV3 - OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot . GAPDH was used as loading control ( E , F ) Signalling kinetics are presented for pAXL and pERK in SKOV3 - EMPTY " E " and SKOV3 - OCPML " O " , treated with MBCD and no treatment control , normalised to GAPDH , from ( D ) ( G ) PLA assay of AXL - OPCML interaction ( red ) upon Gas6 stimulation in both MBCD treated and non - treated SKOV3 - OPCML cells . Scale bar =

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

Figure 5 (A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium(B) Western blotting of SKOV3-OPCML cells treated with MBCD and separated into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation(C) Ratio of AXL band intensity in "R" or pAXL band intensity in "S" relative to total AXL "T" from SKOV3-OPCML "O" in (B)(D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control(E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D)(G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm


Figure 7 ( B ) Western Blotting of SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells pre incubated with either no drug ( DMSO only control ) , 1 . 09 μM R428 , or 2 . 84 μM R428 for an hour and stimulated with Gas6 for the indicated times . GAPDH was used as loading control ( C ) Immunofluorescence staining of pERK ( red ) and OPCML ( magenta ) in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with R428 and stimulated with Gas6 for the indicted time points . Scale bars =

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

Figure 7(B) Western Blotting of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control(C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm


Figure 1D Western blot of Sec63 , mCherry and Pgk1 from WT cells ( SSY1404 ) , cells harboring the estradiol - inducible system ( SSY1405 ) or ∆ opi1 cells ( SSY1607 ) , all of which expressed Sec63 - mNeon and Rtn1 - mCherry . Cells were untreated or treated with 800 nM estradiol for 6 h . Pgk1 served as a loading control . E Quantification of ER size in estradiol - treated cells harboring the inducible system ( SSY1405 ) , untreated ∆ opi1 cells ( SSY1607 ) and WT cells ( SSY1404 ) treated with 8 mM DTT for 1 h . Bars represent mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with 0 nM estradiol or ∆ opi1 cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 ; n . s . , not significant .

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Figure 1D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control.E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.


Figure 2A Sec63 - mNeon images of cells of the indicated genotypes harboring the inducible system . Cells were treated with 800 nM estradiol for 6 h . Two examples of each phenotypic class are shown . C Sec63 - mNeon and Rtn1 - mCherry images of ∆ lnp1 and ∆ sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h .

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Figure 2A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol for 6 h. Two examples of each phenotypic class are shown.C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1 and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.


Figure 3C Quantification of peripheral ER structures in WT and ∆ ice2 cells harboring the inducible system ( SSY1405 , 1603 ) and treated with 800 nM estradiol for the times indicated . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant . E Quantification of peripheral ER structures in untreated WT , Δice2 , Δopi1 and Δice2 Δopi1 cells ( SSY1404 , 2356 , 2595 , 2811 ) . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

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Figure 3C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


Figure 4C , D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63 - mNeon and Rtn1 - mCherry ( SSY1405 , 1603 ) that were untreated ( C ) or treated with 8 mM DTT for 1 h ( D ) .

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Figure 4C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).


Figure 5B Growth assays of untreated WT , Δice2 , Δnem1 , Δnem1 Δice2 , Δspo7 , Δspo7 Δice2 , Δpah1 and Δpah1 Δice2 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 , 2807 , 2808 ) . Numbers represent areas under the curves and serve as growth indices . Mean + s . e . m . , n = 3 biological replicates . Data for WT and ∆ ice2 cells are the same in the left and middle panels . C , D Lipidomic analysis of WT , Δice2 , Δnem1 , Δice2 Δnem1 , Δspo7 and Δice2 Δspo7 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 . Data for WT and Δice2 cells are the same as in both panels . E Sec63 - mNeon images of untreated WT , Δnem1 , Δnem1Δice2 , Δspo7 and Δspo7 Δice2 cells ( SSY1404 , 2482 , 2484 , 2481 , 2483 ) .

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Figure 5B Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.C, D Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).


Figure 6D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1 - HA from ∆ nem1 mutants ( SSY3065 ) and microsomes from cells of the indicated genotypes ( SSY3053 , 3074 , 3075 , 3095 ) . Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase ( PPase ) are shown for reference . E Western blot of HA , Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ ice2 cells expressing Nem1 - HA ( SSY3140 , 3141 ) . Nem1 is undetectable in cell lysates due to its low abundance .

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Figure 6D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.


Figure 7Western blots of FLAG , HA and Dpm1 from cell lysates or anti - FLAG immunoprecipitates of WT or ∆ nem1 cells containing Spo7 - FLAG , Nem1 - FLAG or Ice2 - HA as indicated ( SSY122 , 2421 , 3183 , 3184 , 3195 , 3196 , 3197 ) . The asterisk marks the position of the light chain from the antibody used for immunoprecipitation . The diamond marks a protein that is non - specifically precipitated by the anti - FLAG antibody . IP , immunoprecipitate . E Images of cells with endogenously tagged Ice2 - mScarlet and Spo7 - mNeon or Nem1 - mNeon ( SSY3244 , 3245 ) . Arrows indicate foci containg both Ice2 and either Spo7 or Nem1 . F Quantification of Spo7 and Nem1 foci in WT and ∆ ice2 cells ( SSY2916 , 3238 , 2917 , 3239 ) . Mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with the respective WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 .

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Figure 7Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, Nem1-FLAG or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197). The asterisk marks the position of the light chain from the antibody used for immunoprecipitation. The diamond marks a protein that is non-specifically precipitated by the anti-FLAG antibody. IP, immunoprecipitate.E Images of cells with endogenously tagged Ice2-mScarlet and Spo7-mNeon or Nem1-mNeon (SSY3244, 3245). Arrows indicate foci containg both Ice2 and either Spo7 or Nem1. F Quantification of Spo7 and Nem1 foci in WT and ∆ice2 cells (SSY2916, 3238, 2917, 3239). Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the respective WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


Figure 8A Lipidomic analysis of WT and ∆ ice2 cells in which PAH1 was replaced with PAH1 - HA or pah1 ( 7A ) - HA as indicated ( SSY2841 , SSY2842 , SSY2970 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 .

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Figure 8A Lipidomic analysis of WT and ∆ice2 cells in which PAH1 was replaced with PAH1-HA or pah1(7A)-HA as indicated (SSY2841, SSY2842, SSY2970). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


Figure 9A Sec63 - mNeon images of mid and cortical sections of untreated WT and ∆ opi1 cells , overexpressing ICE2 where indicated ( SSY1404 , 2588 , 2595 , 2596 ) . D Growth assays on solid media of WT , ∆ hac1 , Δice2 , and Δhac1 Δice2 cells ( SSY1404 , 2356 , 2805 , 2806 ) in the absence or presence of 0 . 2 µg / ml tunicamycin . For each series , cells were diluted fivefold from one step to the next .

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Figure 9A Sec63-mNeon images of mid and cortical sections of untreated WT and ∆opi1 cells, overexpressing ICE2 where indicated (SSY1404, 2588, 2595, 2596).D Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.


Figure 2 ( A - F ) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA ( A ) , surface TRPV1 ( B ) , Rab3A ( C ) , synaptotagmin ( D ) , DENN / MADD ( E ) and α - tubulin ( F ) . TrkA and TRPV1 were visualized without permeabilization . ns , p > 0 . 05 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Welch ' s t test from at least three independent cultures . Error bars show SEM . Scale bars , 2 µm .

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Figure 2(A-F) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA (A), surface TRPV1 (B), Rab3A (C), synaptotagmin (D), DENN/MADD (E) and α-tubulin (F). TrkA and TRPV1 were visualized without permeabilization. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Welch's t test from at least three independent cultures. Error bars show SEM. Scale bars, 2 µm.


Figure 3Time - lapse fluorescence images of DRG neurons expressing KIF1A - EGFP and KIF1A ( E239K ) - EGFP ( B ) Arrowheads in ( B ) show the anterogradely moving particles . Scale bar in ( B ) , 2 μm . See Movies EV1 and 2 . ( F ) Movements of KIF1A - EGFP and KIF1A ( E239K ) - EGFP expressed in KIF1A knockdown DRG and the scrambled control . White scale bar , 1 μm . ( G ) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes . Data are shown as mean and SD . KD + KIF1A - EGFP , n = 29 ; KD + KIF1A ( E239K ) - EGFP , n = 32 ; scrambled + KIF1A - EGFP , n = 44 ; scrambled + KIF1A ( E239K ) - EGFP , n = 42 from three independent cultures . ns , p = 0 . 3329 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Unpaired t test .

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Figure 3Time-lapse fluorescence images of DRG neurons expressing KIF1A-EGFP and KIF1A(E239K)-EGFP (B) Arrowheads in (B) show the anterogradely moving particles. Scale bar in (B), 2 μm. See Movies EV1 and 2.(F) Movements of KIF1A-EGFP and KIF1A(E239K)-EGFP expressed in KIF1A knockdown DRG and the scrambled control. White scale bar, 1 μm. (G) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes. Data are shown as mean and SD. KD+KIF1A-EGFP, n = 29; KD+KIF1A(E239K)-EGFP, n = 32; scrambled+KIF1A-EGFP, n = 44; scrambled+KIF1A(E239K)-EGFP, n = 42 from three independent cultures. ns, p = 0.3329; **, p < 0.01; ****, p < 0.0001; Unpaired t test.


Figure 4 ( A ) Representative kymographs of tetramethylrhodamine - labelled microtubule movement driven by the WT and the E239K mutant . The arrowheads represent the end positions of the WT and E239K . ( D ) ATPase activity of WT ( green ) and E239K mutant ( magenta ) activated by microtubules . Data are represented as the mean ± SD . The data from at least three independent experiments were analysed . * * * * , p < 0 . 0001 , two - way ANOVA . ( F ) Rate constants ( kobs ) determined for the reaction of mant - ATP with nucleotide - free KIF1A ( WT ) and KIF1A ( E239K ) were plotted against concentration of mant - ATP . Data are represented as the mean ± SD . The data from three independent measurements were analysed .

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Figure 4(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K.(D) ATPase activity of WT (green) and E239K mutant (magenta) activated by microtubules. Data are represented as the mean ± SD. The data from at least three independent experiments were analysed. ****, p < 0.0001, two-way ANOVA.(F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed.


Figure 6 ( A ) The positions of the β6 - L10 - β7 and neck - coil peptides are shown in brown and green . The positions of the neck - linker peripheral peptides ( α1 - β3 - P loop , α4 - L12 and L12 - α5 ) are shown in light blue . ( B - D ) Quantitation of site - specific intramolecular interactions . Representative MS / MS spectra of the indicated peptides , including the mutated amino acid ( β6 - L10 - β7 ) ( B and C ) and neck - coil ( D ) . In the inserted graphs , the intensities in the ADP - BeFx and ADP - AlFx states were measured with respect to the nucleotide - free state as 1 . 0 . Grey bar graphs , WT ; black bar graphs , E239K ; ( - ) , nucleotide - free .

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Figure 6(A) The positions of the β6-L10-β7 and neck-coil peptides are shown in brown and green. The positions of the neck-linker peripheral peptides (α1-β3-P loop, α4-L12 and L12-α5) are shown in light blue. (B-D) Quantitation of site-specific intramolecular interactions. Representative MS/MS spectra of the indicated peptides, including the mutated amino acid (β6-L10-β7) (B and C) and neck-coil (D). In the inserted graphs, the intensities in the ADP-BeFx and ADP-AlFx states were measured with respect to the nucleotide-free state as 1.0. Grey bar graphs, WT; black bar graphs, E239K; (-), nucleotide-free.


Figure 1 . ( e ) Binding of monoclonal antibodies to 2019 - nCoV RBD determined by ELISA . ( f ) Binding profiles of 2019 - nCoV RBD to ACE2 and antibodies ( g ) competition of CR3022 and ACE2 with 2019 - nCoV RBD measured by BLI in OctetRED96 . Binding kinetics was evaluated using a 1 : 1 Langmuir binding model by ForteBio Data Analysis 7 . 0 software .

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Figure 1.(e) Binding of monoclonal antibodies to 2019-nCoV RBD determined by ELISA.(f) Binding profiles of 2019-nCoV RBD to ACE2 and antibodies(g) competition of CR3022 and ACE2 with 2019-nCoV RBD measured by BLI in OctetRED96. Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.


Figure 4C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6 ' s affinity towards H3K9me2 . Exemplary IF microscopy images of the indicated fission yeast strains are shown . Homothallic strains were incubated on SPAS plates . DNA and tubulin were stained with DAPI and anti - TAT - 1 , respectively . Missegregating chromosome is indicated with a yellow arrow ; D Quantification of meiotic chromosome segregation phenotypes . The data for clr4 + and clr4F449Y is the same as in Fig 3B . P values were calculated using Fisher ' s exact test . Number of total cells counted is indicated in each bar . E Comparison of co - segregation of centromere I ( CenI ) during MI in cells lacking H3K9me3 depending on Swi6 ' s affinity towards H3K9me2 . Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown . Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis . Heterozygous CenI - GFP was used to follow the segregation pattern during meiosis . The GFP - signal is additionally displayed in greyscale for better visibility F Quantification of CenI - GFP segregation during meiosis . The data for clr4 + and clr4F449Y is the same as in Fig 3D . P values were calculated using Fisher ' s exact test . Number of total cells counted is indicated in each bar . G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1 - like - CD cells . Exemplary image ( left ) and quantification of the spore viability ( right ) are shown . Meiosis was induced in homothallic cells and the resulting spores were dissected . The data for clr4 + and clr4F449Y is the same as in Fig 3F . P values were calculated using Fisher ' s exact test . Number of dissected tetrads is indicated in each bar .

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 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

Figure 4C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary IF microscopy images of the indicated fission yeast strains are shown. Homothallic strains were incubated on SPAS plates. DNA and tubulin were stained with DAPI and anti-TAT-1, respectively. Missegregating chromosome is indicated with a yellow arrow; D Quantification of meiotic chromosome segregation phenotypes. The data for clr4+ and clr4F449Y is the same as in Fig 3B. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.E Comparison of co-segregation of centromere I (CenI) during MI in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown. Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis. Heterozygous CenI-GFP was used to follow the segregation pattern during meiosis. The GFP-signal is additionally displayed in greyscale for better visibility F Quantification of CenI-GFP segregation during meiosis. The data for clr4+ and clr4F449Y is the same as in Fig 3D. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1-like-CD cells. Exemplary image (left) and quantification of the spore viability (right) are shown. Meiosis was induced in homothallic cells and the resulting spores were dissected. The data for clr4+ and clr4F449Y is the same as in Fig 3F. P values were calculated using Fisher's exact test. Number of dissected tetrads is indicated in each bar.


Figure 6A Western blot of whole - cell lysates ( WCL ) using a PhosTag gel for anti - FLAG ( top ) and a Bis - Tris gel for anti - Act1 detection ( bottom ) , respectively . WCL were prepared from mitotic and meiotic 3xFLAG - clr4 + / 3xFLAG - clr4 + cultures at the indicated time points . C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains . D Western blot of FLAG IPs in 3xFLAG - clr4 + cdc2 - as cells , treated with 2 µM 1 - NM - PP1 for the indicated amount of time . The blot was first probed with the phospho - specific anti - pS458 antibody , stripped , and reprobed with an anti - FLAG antibody .

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

Figure 6A Western blot of whole-cell lysates (WCL) using a PhosTag gel for anti-FLAG (top) and a Bis-Tris gel for anti-Act1 detection (bottom), respectively. WCL were prepared from mitotic and meiotic 3xFLAG-clr4+/3xFLAG-clr4+ cultures at the indicated time points.C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains.D Western blot of FLAG IPs in 3xFLAG-clr4+ cdc2-as cells, treated with 2 µM 1-NM-PP1 for the indicated amount of time. The blot was first probed with the phospho-specific anti-pS458 antibody, stripped, and reprobed with an anti-FLAG antibody.