Figure 2Two human ( SKMEL28 and 1205Lu ) and a murine ( B16BL6 ) melanoma cell lines were exposed , or not , to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) , then FAO was measured ( n = 5 for B16BL6 , SKMEL28 HFD EV and 1205Lu ND EV and n = 6 for other conditions ) . Volcano plot of mass spectrometry - based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice ( HFD ) , as compared to those from lean mice ( ND ) . The dashed lines indicate cut - off values and points colored in grey indicate proteins that display non - significant fold - change by Welch t - test between both conditions ( n = 3 for ND , 4 for HFD ) . Proteins involved in FAO are indicated by yellow dots . Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice ( top panel ) and from human individuals with varying BMI ( normal weight , NW ; overweight , OW and obese , OB ) ( bottom panel ) . For each blot , extracts from 3 independent batches of murine samples or 3 independent individuals for human samples ( 1 - 3 ) are shown . Flotillin 1 ( FLOT1 ) is used as a loading control . Western blot analysis of the indicated FAO enzymes in melanoma cells treated , or not , with EV from lean ( ND ) and obese ( HFD ) mice . Tubulin ( TUB ) is used as a loading control . RT - qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice for 48 h . Results are expressed relative to the corresponding value for control cells ( n = 3 ) . Analysis of FAO levels in 1205Lu cells exposed to 3T3 - F442A EV and treated , or not , with cycloheximide ( CHX ) ( n = 5 ) .

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Figure 2Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions).Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cut-off values and points colored in grey indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n=3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots.Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW and obese, OB) (bottom panel). For each blot, extracts from 3 independent batches of murine samples or 3 independent individuals for human samples (1-3) are shown. Flotillin 1 (FLOT1) is used as a loading control.Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control.RT-qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n=3).Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).


Figure 3Lipids were extracted from EV secreted by 3T3 - F442A pre - adipocytes and differentiated 3T3 - F442A adipocytes ( respectively preAd - 3T3 - EV and Ad - 3T3 - EV ) , and FA content was measured ( n = 6 ) . Indicated melanoma cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 ( 3T3 - FL C16 - EV ) and , 24h later , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy . Left panel , indicated cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated , or not , with Etomoxir for 24h . Then , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy . Right panel , quantification of BODIPY FL C16 staining area per cell ( n = 3 ) . Lipids were extracted from EV secreted by adipocytes from lean ( ND ) and obese ( HFD ) mice ( n = 5 ) ( left panel ) or from human adipose tissue samples from patients with varying BMI ( right panel ) and fatty acid content was measured ( n = 15 ) . Indicated cells were exposed , or not , to adipocyte EV from primary adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) for 24h . Then , cells were fixed , stained with BODIPY and counterstained with DAPI . Left panel , confocal microscopy observation . Right panel , quantification of BODIPY staining area per cell ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

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Figure 3Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy.Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3).Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15).Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).


Figure 4Transmission electron micrographs of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) . Scale bar represents 1µm . A zoomed crop of the area with autophagic structures containing lipids ( indicated by an arrow ) is shown ( scale bar : 0 . 5µm ) . 1205Lu cells were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 in the presence , or not , of Lalistat 2 . Then , live cells were stained with the LysoTracker probe and observed by confocal microscopy . Arrows indicate colocalization . 1205Lu were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 and treated , or not , with Lalistat 2 . Then , cells were fixed and counterstained with DAPI before observation by confocal microscopy . Quantification of BOBIPY FL C16 staining per cell is shown beside ( n = 4 ) . 1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Lalistat 2 ( Lal ) . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 5 ) or cell motility was tracked by video microscopy ( right panel ; n = 40 cells per group ) . 1205Lu cells were exposed , or not , to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) with , or without , Lalistat 2 ( Lal ) . Cells were then fixed , stained with BODIPY and counterstain with DAPI before observation by confocal microscopy . Quantification of BODIPY staining per cell is shown on the right ( n = 5 ) . 1205Lu cells were exposed , or not , to adipocyte EV from ND or HFD mice with , or without , Lalistat 2 ( Lal ) . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

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Figure 4Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm).1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization.1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4).1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group).1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5).1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).


Figure 51205Lu cells exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 , were stained with a MitoTracker probe , fixed and observed by confocal microscopy . Arrows indicate the presence of mitochondria in membrane protrusions . On the images to the right , the outline of cells is shown in dotted lines . Scale bars : 20µm . Right panel , quantification of the distance between mitochondria and nuclei ( n = 15 ) . Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated , or not , with EV from 3T3 - F442A adipocytes ( 3T3 - EV ) . Tubulin ( TUB ) is used as a loading control . 1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 6 ) . Right panel , 3T3 - F442A EV were added to 1205Lu cells , which were immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) ( right panel ) . 1205Lu cells were transfected , or not , with 2 different siRNA targeted against DRP1 ( siDRP1 # 1 or 2 ) or an untargeted siRNA ( siUT ) . Top , 48h after transfection , protein extracts were prepared and DRP1 expression was evaluated by western blot . Bottom , 36h post transfection , cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and cell motility was tracked by video microscopy ( n = 40 cells per group ) . 1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) and immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

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Figure 51205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control.1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group).1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).


Figure 6Left panel , indicated melanoma cells exposed to 3T3 - F442A EV were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 6 ) . Left panel , melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) . 1205Lu cells exposed to 3T3 - F442A EV were stained with a MitoTracker probe . Then , cells were fixed and stained with BODIPY before observation by confocal microscopy . A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown ( scale bar : 5µm ) . Transmission electron microscope observations of 1205Lu cells exposed , or not , to 3T3 - F442A EV . Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right . Number of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) ( n = 28 for control and n = 27 for 3T3 - EV ) . Area of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( n = 19 for control and n = 25 for 3T3 - EV ) . Left panel , 1205Lu melanoma cells were exposed , or not , to 3T3 - F442A EV . Then , live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy . Right panel , quantification of the percentage of cells presenting lysosome within membrane protrusions ( n = 3 ) .

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Figure 6Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6).Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu).1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm).Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right.Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV) (n=28 for control and n=27 for 3T3-EV). Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (n=19 for control and n=25 for 3T3-EV).Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).


Figure 7TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 449 , 450 and 459 for HADHA , HADHB and ECHS1 respectively ) . BODIPY staining of neutral lipid stores in the indicated melanoma cell lines , which present increasing degrees of aggressiveness ( from left to right ) . DAPI was used to counterstain nuclei . Scale bar : 20µm . FAO in the indicated melanoma cell lines was measured . Bars and error bars represent means ± SEM ( n = 5 ) ; statistically significant by one - way ANOVA with post hoc Tukey ' s test , * * * P < 0 . 001 . If not indicated , non - significant . TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 459 ) .

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Figure 7TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=449, 450 and 459 for HADHA, HADHB and ECHS1 respectively).BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.FAO in the indicated melanoma cell lines was measured. Bars and error bars represent means ± SEM (n=5); statistically significant by one-way ANOVA with post hoc Tukey's test, ***P < 0.001. If not indicated, non-significant.TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=459).


Figure 1A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 and tubulin were used as loading controls . * non ‐ specific band . B Cells treated as in ( A ) were fixed 48 h after selection and stained with an anti ‐ 53BP1 antibody ( green ) . Telomeres were visualized with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( red ) . Yellow arrowheads point to 53BP1 foci that co ‐ localize with telomeres . C Quantification of TIFs in cells treated as in ( B ) . A minimum of 200 nuclei were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 .

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Figure 1A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band.B Cells treated as in (A) were fixed 48 h after selection and stained with an anti‐53BP1 antibody (green). Telomeres were visualized with a Cy3‐conjugated (CCCTAA)3‐PNA probe (red). Yellow arrowheads point to 53BP1 foci that co‐localize with telomeres.C Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05.


Figure 2A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO ‐ FISH analysis . Metaphase chromosome spreads were stained with Cy3 ‐ conjugated leading strand telomeric PNA probe ( red ) and FITC ‐ conjugated lagging strand telomeric PNA probe ( green ) . DNA was counter ‐ stained with DAPI ( blue ) . B The frequency of chromosome ‐ type telomeric fusions in cells treated as in ( A ) was quantified as a percentage of total number of chromosomes ( illustrated in Supplementary Fig S2A ) . A minimum of 2 , 000 chromosomes were scored for each treatment . Error bars represent SD of at least two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; NS , P > 0 . 05 .

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Figure 2A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).B The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.


Figure 3A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 . B Immortalized Brca1F / C61G MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . The frequency of end ‐ to ‐ end chromosome ‐ type fusions was analysed as in ( A ) . C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . GAPDH was used as a loading control . * non ‐ specific band . C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cells treated as in ( C ) and ( E ) were arrested in mitosis with colcemid , and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( D , F ) . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 . C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . GAPDH was used as a loading control . * non ‐ specific band . C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cells treated as in ( C ) and ( E ) were arrested in mitosis with colcemid , and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( D , F ) . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 .

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 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 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 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

Figure 3A Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.B Immortalized Brca1F/C61G MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. The frequency of end‐to‐end chromosome‐type fusions was analysed as in (A).C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.


Figure 4A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 was used as a loading control . * non ‐ specific band . B Quantification of the frequency of end ‐ to ‐ end chromosome ‐ type fusions of cells treated as in ( A ) represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . The P ‐ value was calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 . Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 . D Immortalized Brca1F / − MEFs were infected with retroviruses expressing TRF2 shRNAs and / or Cre recombinase , together with a lentivirus expressing PARP1 shRNA , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 was used as a loading control . * non ‐ specific band . E Quantification of the frequency of end ‐ to ‐ end chromosome ‐ type fusions of cells treated as in ( D ) represented as a percentage of fusions observed after TRF2 depletion . A minimum of 1 , 500 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . The P ‐ value was calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

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

Figure 4A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.B Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (A) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


Figure 5A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 and GAPDH were used as loading controls . * non ‐ specific band . B MboI ‐ and AluI ‐ digested DNA from cells treated as in ( A ) was resolved by pulsed ‐ field gel electrophoresis and probed with end ‐ labelled ( AACCCT ) 4 probe . Representative pulsed ‐ field gel samples run under native and denatured conditions are shown . C Quantification of the 3 ′ overhang in cells treated as in ( B ) . For each sample , the ss / total DNA ratios were expressed relative to the GFP shRNA ‐ treated control . Error bars represent SD of two independent experiments .

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

Figure 5A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and GAPDH were used as loading controls. *non‐specific band.B MboI‐ and AluI‐digested DNA from cells treated as in (A) was resolved by pulsed‐field gel electrophoresis and probed with end‐labelled (AACCCT)4 probe. Representative pulsed‐field gel samples run under native and denatured conditions are shown.C Quantification of the 3′ overhang in cells treated as in (B). For each sample, the ss/total DNA ratios were expressed relative to the GFP shRNA‐treated control. Error bars represent SD of two independent experiments.


Figure 6A - C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment . A minimum of 1 , 200 chromosomes were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 . Each graph represents a separate set of experiments . A - C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment . A minimum of 1 , 200 chromosomes were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 . Each graph represents a separate set of experiments . A - C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment . A minimum of 1 , 200 chromosomes were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 . Each graph represents a separate set of experiments .

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 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 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

Figure 6A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.


Figure 1 ( C - G ) Resembling the phenotype caused by Notch overexpression ( D ) or dSir2 mutation ( E ) , HP1c knockdown ( F ) and mutant ( G ) flies also exhibit supernumerary anterior scutellar ( aSC ) bristles on the notum , as indicated by the enlarged views in the left bottom panes . Scale bars , 100 μm . ( H - M ) Compared with control flies ( H and K ) , HP1c knockdown ( J ) and mutant ( M ) flies exhibit an ectopic vein at the distal of L4 and L5 ( arrows ) and a broken posterior cross vein ( arrowhead ) , which is similar to the phenotype caused by Notch overexpression ( I ) and dSir2 mutant ( L ) . Scale bars , 500 μm .

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

Figure 1(C-G) Resembling the phenotype caused by Notch overexpression (D) or dSir2 mutation (E), HP1c knockdown (F) and mutant (G) flies also exhibit supernumerary anterior scutellar (aSC) bristles on the notum, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm.(H-M) Compared with control flies (H and K), HP1c knockdown (J) and mutant (M) flies exhibit an ectopic vein at the distal of L4 and L5 (arrows) and a broken posterior cross vein (arrowhead), which is similar to the phenotype caused by Notch overexpression (I) and dSir2 mutant (L). Scale bars, 500 μm.


Figure 2 ( A - D ) The posterior midguts of 10 - day - old control ( A ) , HP1c knockdown ( B ) , HP1c null mutant ( C ) and HP1c overexpression flies ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - EB , EB - EB or ISC - ISC nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm . ( E - I ) Confocal images of the posterior midgut clones of the indicated genotypes , marked by GFP ( dotted lines ) . Arrows mark ISCs / EBs , arrowheads mark ee cells , and asterisks mark ECs . DNA was visualized with DAPI and ee cells were stained with Pros . Scale bars , 10 μm .

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

Figure 2(A-D) The posterior midguts of 10-day-old control (A), HP1c knockdown (B), HP1c null mutant (C) and HP1c overexpression flies (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-EB, EB-EB or ISC-ISC nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.(E-I) Confocal images of the posterior midgut clones of the indicated genotypes, marked by GFP (dotted lines). Arrows mark ISCs/EBs, arrowheads mark ee cells, and asterisks mark ECs. DNA was visualized with DAPI and ee cells were stained with Pros. Scale bars, 10 μm.


Figure 3 ( A - D ) Compared with the control ( A ) flies , posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells ( B ) , while HP1c OE flies show increased ISCs and ee cells ( C ) , and the phenotype of Notch OE can be rescued by HP1c OE ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - ISC , ISC - EB or EB - EB nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


Figure 4ChIP - qPCR results show the association of HP1c with Notch target genes compared to RFP control . The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin ( equivalent to 1 ) . Error bars indicate SEM . n = 3 technical replicates . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . ( B ) RT - qPCR analysis determined the expression of Notch target genes when HP1c was depleted ( n = 3 technical replicates , , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) . NRE - GFP signals of S2 cells carrying NICD OE , Hairless KD , Su ( H ) KD , dSir2 KD and HP1c KD , and the statistical results ( F ) ( n = 4 biological replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * 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 O O O O O O O O O O O O O O O O O O O O O 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

Figure 4ChIP-qPCR results show the association of HP1c with Notch target genes compared to RFP control. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).(B) RT-qPCR analysis determined the expression of Notch target genes when HP1c was depleted (n=3 technical replicates,, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, ***p < 0.001).NRE-GFP signals of S2 cells carrying NICD OE, Hairless KD, Su(H) KD, dSir2 KD and HP1c KD , and the statistical results (F) (n=4 biological replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


Figure 5 ( D , E ) The interaction between HP1c and Su ( H ) is further confirmed through co - immunoprecipitation ( Co - IP ) . Plasmids expressing HP1c - eGFP and Su ( H ) - flag were transfected into S2 cells . Immunoprecipitation and western blotting were performed with the indicated antibodies .

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

Figure 5(D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.


Figure 6 ( B ) The relative binding of HP1c with Notch target genes when Su ( H ) KD using ChIP - qPCR analysis ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) The relative binding of HP1c on Notch target genes when NICD OE using ChIP - qPCR analysis . ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * * * 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 O O O O O O O O O O O O O O O O

Figure 6(B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01). (C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001).


Figure 7 ( M ) Statistical results of the number of ISCs and ee cells per unit area ( 200 μm × 200 μm ) in ( D - F ) . More than twenty - five midguts were examined for each group . Error bars indicate SEM . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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 7(M) Statistical results of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in (D-F). More than twenty-five midguts were examined for each group. Error bars indicate SEM. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).


Figure 1A HeLa cells expressing LAMP1 - RFP were treated with vehicle alone ( control ) or LLOMe for 1 h to induce lysosome damage . Cells were fixed 2 h after washout , stained with p62 and galectin - 3 ( Gal3 ) antibodies , and analyzed by confocal microscopy . B p97 translocates to ruptured lysosomes . Cells co - expressing LAMP1 - RFP and GFP - p97 were treated as in ( A ) and stained with the pan - ubiquitin antibody FK2 ( Ub ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) . C Immunodetection of endogenous p97 after LLOMe treatment as in ( A ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) . D Clearance of damaged lysosomes depends on p97 . Cells were transfected with control ( Ctrl ) or p97 siRNA oligos , fixed at indicated time points after LLOMe washout and stained for endogenous Gal3 . Quantification of the number of Gal3 - positive vesicles per cell . Data represent mean ± SD of four independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s unequal variances t - tests comparing the siCtrl with sip97 ) . E Cells were chased after LLOMe treatment for 12 h with vehicle alone ( DMSO ) , bortezomib ( Btz , 10 nM ) , NH4Cl ( 20 mM ) or the p97 inhibitor NMS - 873 ( 5 µM ) , and Gal3 vesicles quantified . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s t - tests ) . F p97 is essential for cell survival after lysosome rupture . Cells were transfected with control ( Ctrl ) , p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h . Cell viability was measured using the MTS assay . Data represent mean ± SD of three independent experiments . G Taufibrils are endocytosed and induce lysosomal damage . Taufibrils were generated in vitro , fluorescently labelled by Dylight488 and fragmented by sonication ( 488 - labelled tau ) . HeLa cells expressing mCherry - Gal3 were incubated with 300 nM taufibrils for 24 h . Arrows indicate compartments that contain 488 - labelled tau and are positive for mCherry - Gal3 indicating lysosome rupture . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) . H Accumulation of tau - induced lysosomal damage upon p97 depletion . Cells transfected with control ( Ctrl ) or p97 siRNA oligos were treated as in ( G ) . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 . Scale bars , 10 µm .

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

Figure 1A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy.B p97 translocates to ruptured lysosomes. Cells co-expressing LAMP1-RFP and GFP-p97 were treated as in (A) and stained with the pan-ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97).E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests).F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.G Taufibrils are endocytosed and induce lysosomal damage. Taufibrils were generated in vitro, fluorescently labelled by Dylight488 and fragmented by sonication (488-labelled tau). HeLa cells expressing mCherry-Gal3 were incubated with 300 nM taufibrils for 24 h. Arrows indicate compartments that contain 488-labelled tau and are positive for mCherry-Gal3 indicating lysosome rupture. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD).H Accumulation of tau-induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Scale bars, 10 µm.


Figure 2A Impaired clearance of damaged lysosomes in cells expressing a p97 disease - mutant . Wild - type and p97R155H / + MEFs were LLOMe - treated for 1 h and released for indicated times . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test ) . Scale bar , 25 µm . B Stable U2OS cell lines were doxycycline - induced to express myc - tagged p97 wild - type or disease - mutants as indicated . Induced parental cells served as control ( U2OS - ) . Cells were treated with LLOMe for 1h and chased for indicated time before fixation . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments . Comparable expression levels were verified ( see Figure EV2A ) . C Stable U2OS cell lines described in ( B ) were treated with LLOMe for 1 h and chased for indicated time . LC3 and GAPDH levels were analyzed by immunoblotting . Quantification is shown in Figure EV2B . D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle . Patient biopsies immunostained for Gal3 and caveolin - 3 ( Cav3 ) . Quantification from 4 random 10X fields for each biopsy and means for controls and patients , respectively . Patients carry p97 mutations R155H ( # 1 - 3 ) or R93C for ( # 4 ) . Patients # 2 and # 3 are cousins . Scale bar , 100 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 2A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student's unpaired t-test). Scale bar, 25 µm.B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A).C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B.D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle. Patient biopsies immunostained for Gal3 and caveolin-3 (Cav3). Quantification from 4 random 10X fields for each biopsy and means for controls and patients, respectively. Patients carry p97 mutations R155H (#1-3) or R93C for (#4). Patients #2 and #3 are cousins. Scale bar, 100 µm.


Figure 3A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes . HeLa cells were transfected with indicated siRNA oligos and LLOMe - treated for 1 h . Cells were chased for 12 h , fixed and stained for Gal3 . See Figure EV3 for depletion efficiency . B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition . This threshold was chosen so that less than 1 % of untreated cells were considered positive . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - sided Welch ' s t - tests ) . C YOD1 , UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes . Cells expressing indicated tagged proteins were LLOMe - treated for 1 h or with vehicle alone ( control ) , fixed 2 h after washout , and stained for endogenous LAMP1 and , in the case of PLAA , with Alexa - 488 conjugated anti - HA antibodies . See Figure EV3B for quantification . D An ATP - stabilized complex of p97 containing both UBXD1 and PLAA . Stable HEK293 cell lines were doxycycline - induced to express p97 wild - type ( wt ) or the ATPase - mutant E578Q ( EQ ) at near endogenous level . Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies . Arrowheads indicate endogenous ( lower band ) and induced p97 ( upper band ) . E Stable HEK293 cells treated as in ( D ) and transiently expressing GFP - tagged YOD1 - wt or the YOD1 - C160S catalytic mutant ( CS ) were processed for co - immunoprecipitation with GFP nanobodies . Asterisk , unspecific band . Note that the YOD1 - CS mutant stabilized p97 binding and , in the p97 - EQ background , also associated with UBXD1 and PLAA . F Control ( IgG ) and UBXD1co - immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP - YOD1 - CS after formaldehyde cross - linking . Ufd1 served as a negative control . Scale bars , 10 µm .

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

Figure 3A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes. HeLa cells were transfected with indicated siRNA oligos and LLOMe-treated for 1 h. Cells were chased for 12 h, fixed and stained for Gal3. See Figure EV3 for depletion efficiency.B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition. This threshold was chosen so that less than 1% of untreated cells were considered positive. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-sided Welch's t-tests).C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification.D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band).E Stable HEK293 cells treated as in (D) and transiently expressing GFP-tagged YOD1-wt or the YOD1-C160S catalytic mutant (CS) were processed for co-immunoprecipitation with GFP nanobodies. Asterisk, unspecific band. Note that the YOD1-CS mutant stabilized p97 binding and, in the p97-EQ background, also associated with UBXD1 and PLAA.F Control (IgG) and UBXD1co-immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP-YOD1-CS after formaldehyde cross-linking. Ufd1 served as a negative control. Scale bars, 10 µm.


Figure 4A K63 - and K48 - ubiquitination of damaged lysosomes . Untransfected HeLa cells , or cells expressing mCherry - Gal3 ( Ch - Gal3 ) were LLOMe - treated for 1 h . 2 h after washout , cells were stained with antibodies specific for K63 or K48 chains , and LAMP1 or p62 as indicated . Percentage of K63 - or K48 - ubiquitinated LAMP1 vesicles and percentage of p62 - positive K63 or K48 vesicles were quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test for LAMP1 ) . * = p < 0 . 05 . B Colocalization of K63 - and K48 - linked ubiquitin conjugates on individual lysosomes . Cells expressing GFP - TAB2 - NZF ( K63 - sensor ) were treated as in ( A ) and stained for K48 chains and LAMP1 . Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure ( van Wijk et al . , 2012 ) . C Cells expressing GFP - p97 - EQ were treated as in ( A ) and stained with antibodies as indicated . D Quantification of the total number of K63 - or K48 - ubiquitinated vesicles per cell , and number of p97 - EQ positive K63 - or K48 - ubiquitinated vesicles ( mean ± SD ) . E Percentage of GFP - p97 - EQ vesicles positive for K63 or K48 chains was determined ( mean ± SD ) . F ELDR components target K48 - conjugates on lysosomes . Detection of GFP - YOD1 - CS or UBXD1 - GFP alone , or co - transfected with PLAA - HA and GFP - p97 - EQ , 2 h after LLOMe washout and co - stained with indicated antibodies .

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 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 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

Figure 4A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student's unpaired t-test for LAMP1). * = p < 0.05.B Colocalization of K63- and K48-linked ubiquitin conjugates on individual lysosomes. Cells expressing GFP-TAB2-NZF (K63-sensor) were treated as in (A) and stained for K48 chains and LAMP1. Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure (van Wijk et al., 2012).C Cells expressing GFP-p97-EQ were treated as in (A) and stained with antibodies as indicated.D Quantification of the total number of K63- or K48-ubiquitinated vesicles per cell, and number of p97-EQ positive K63- or K48-ubiquitinated vesicles (mean ± SD).E Percentage of GFP-p97-EQ vesicles positive for K63 or K48 chains was determined (mean ± SD).F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies.


Figure 5A Time course of K63 - and K48 - ubiquitination . Control - depleted cells were fixed at indicated time points after LLOMe washout and stained with chain - specific antibodies and LAMP1 . Automated quantification of LAMP1 vesicles positive for K63 or K48 chains . Data represent mean ± SD of three independent experiments . B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes . Experiment and quantification as in ( A ) in cells treated with p97 or YOD1 siRNA oligos . C The p97 - EQ mutant stabilizes K48 - conjugates . Lysosome damage was induced as in ( A ) in cells transfected with GFP - tagged p97 - wt or p97 - EQ . Cells were chased after washout for 12 h and stained for K48 chains . D Quantification of K48 - positive lysosomes 2 h and 12 h after damage in cells treated as in ( A ) . Data represent mean ± SD of three independent experiments . E The catalytically - inactive mutant of YOD1 stabilizes K48 - conjugates . Cells transfected with GFP - tagged YOD1 - wt or the YOD1 - CS were treated and stained as in ( C ) . F Quantification as in ( D ) of cells treated as in ( E ) . * * = p < 0 . 01 * * * = p < 0 . 001 ( Student ' s unpaired t - test ) . ns , not significant . Scale bars , 10 µm .

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 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 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

Figure 5A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.C The p97-EQ mutant stabilizes K48-conjugates. Lysosome damage was induced as in (A) in cells transfected with GFP-tagged p97-wt or p97-EQ. Cells were chased after washout for 12 h and stained for K48 chains.D Quantification of K48-positive lysosomes 2 h and 12 h after damage in cells treated as in (A). Data represent mean ± SD of three independent experiments.E The catalytically-inactive mutant of YOD1 stabilizes K48-conjugates. Cells transfected with GFP-tagged YOD1-wt or the YOD1-CS were treated and stained as in (C).F Quantification as in (D) of cells treated as in (E). ** = p < 0.01 *** = p < 0.001 (Student's unpaired t-test). ns, not significant. Scale bars, 10 µm.


Figure 6A YOD1 can link p97 to ubiquitin . Ubiquitin - GST ( Ub - GST ) or GST alone was incubated with the indicated purified proteins and isolated . Co - isolated proteins ( pulldown ) were detected in SDS - gels . Note binding of p97 and YOD1 to Ub - GST only in combination . B The S2 site of YOD1 is critical for ubiquitin binding . Experiments as in ( A ) with indicated combinations of p97 and YOD1 - wt or the ubiquitin - binding mutant , MutS2 . C Reconstitution of a complex of ELDR components on ubiquitin . Experiments as in ( A ) with combinations of p97 - wt or p97 - EQ , YOD1 , PLAA and UBXD1 as indicated . Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97 - EQ . D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site . LLOMe - treated cells expressing GFP - YOD1 - CS or the double mutant GFP - YOD1 - CS - MutS2 were chased for 2 h and stained for K48 chains and LAMP1 . Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined . E The S2 site is essential for K48 - conjugate removal . GFP - YOD1 - wt or GFP - YOD1 - MutS2 overexpressing cells treated as in ( D ) were stained for K48 chains . Quantification of the number of K48 - positive vesicles per cell . Data represent mean ± SD of three independent experiments . * * = p < 0 . 01 ( Student ' s unpaired t - test ) . Scale bars , 10 µm .

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Figure 6A YOD1 can link p97 to ubiquitin. Ubiquitin-GST (Ub-GST) or GST alone was incubated with the indicated purified proteins and isolated. Co-isolated proteins (pulldown) were detected in SDS-gels. Note binding of p97 and YOD1 to Ub-GST only in combination.B The S2 site of YOD1 is critical for ubiquitin binding. Experiments as in (A) with indicated combinations of p97 and YOD1-wt or the ubiquitin-binding mutant, MutS2.C Reconstitution of a complex of ELDR components on ubiquitin. Experiments as in (A) with combinations of p97-wt or p97-EQ, YOD1, PLAA and UBXD1 as indicated. Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97-EQ.D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined.E The S2 site is essential for K48-conjugate removal. GFP-YOD1-wt or GFP-YOD1-MutS2 overexpressing cells treated as in (D) were stained for K48 chains. Quantification of the number of K48-positive vesicles per cell. Data represent mean ± SD of three independent experiments. ** = p < 0.01 (Student's unpaired t-test). Scale bars, 10 µm.


Figure 7A Largely mutually exclusive localization of LC3 and K48 chains . GFP - LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains . Correlation coefficients were calculated for colocalization of LC3 with K63 or K48 . The whiskers represent 10th and 90th percentile . Points below and above the whiskers are drawn as individual dots . The single line indicates the median . * * * = p < 0 . 001 ( Mann - Whitney - U test ) . B Lipofectamine - coated latex beads damage lysosomes and induce their K48 - ubiquitination . Cells expressing mCherry - Gal3 were incubated with Lipofectamine - coated latex beads for 3 h and stained with K48 chain - specific antibodies . C YOD1 association and dissociation precedes LC3 recruitment . Live cell video microscopy stills of cells co - expressing mCherry - YOD1 - wt and GFP - LC3 after treatment with Lipofectamine - coated latex beads for 30 min at 2 min image intervals . D Stabilization of K48 - conjugates on lysosomes impairs recruitment of LC3 . Cells expressing GFP - YOD1 - CS were treated as in ( A ) and stained for LAMP1 and p62 or LC3 . Percentage of GFP - YOD1 vesicles positive for p62 or LC3 was determined ( mean ± SD ) . E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation . Cells were transfected with control ( Ctrl ) , p97 or YOD1 siRNA oligos , treated with LLOMe , fixed after 12 h chase and stained for endogenous LC3 and K48 chains . Quantification of the number of K48 - positive vesicles per cell and number of LC3 - positive K48 vesicles per cell was determined . Data represent mean ± SD of three independent experiments . Scale bars , 10 µm .

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Figure 7A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test).B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies.C YOD1 association and dissociation precedes LC3 recruitment. Live cell video microscopy stills of cells co-expressing mCherry-YOD1-wt and GFP-LC3 after treatment with Lipofectamine-coated latex beads for 30 min at 2 min image intervals.D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD).E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.


Figure 1b . Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound - induced tumours ( Pap ) compared to fibroblasts from wild - type ( WT ) and inflamed ( InvEE ) back skin ( n = 3 per condition ) . PRSS35 is indicated in red as a top upregulated gene in CAFs .

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Figure 1b. Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound-induced tumours (Pap) compared to fibroblasts from wild-type (WT) and inflamed (InvEE) back skin (n=3 per condition). PRSS35 is indicated in red as a top upregulated gene in CAFs.


Figure 2a . Immunofluorescent images of skin sections from PDGFRα - eGFP reporter mice with normal ( WT , n = 4 ) , inflamed ( InvEE , n = 4 ) , wounded ( d14 post - wounding ; wound , n = 4 ) , or tumour - associated ( Papilloma , Pap d17 and d30 post - wounding , n = 4 per condition ) skin . Nuclei were stained with Dapi ( 4 ' , 6 - diamidino - 2 - phenylindole ; blue ) . Dotted line represents epidermal - dermal boundary . Scale bars : 50 μm . b . Quantification of the number of fibroblasts present in different skin conditions ( n = 4 mice per condition ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; One - way ANOVA testing ) . Data represent means of multiple microscopic fields ± S . E . M . c . Herovici staining of sections of normal ( WT ) , inflamed ( InvEE ) , wound , or tumour - associated ( Papilloma ) skin . Scale bars : 100 μm .

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Figure 2a. Immunofluorescent images of skin sections from PDGFRα-eGFP reporter mice with normal (WT, n=4), inflamed (InvEE, n=4), wounded (d14 post-wounding; wound, n=4), or tumour-associated (Papilloma, Pap d17 and d30 post-wounding, n=4 per condition) skin. Nuclei were stained with Dapi (4', 6-diamidino- 2- phenylindole; blue). Dotted line represents epidermal-dermal boundary. Scale bars: 50 μm. b. Quantification of the number of fibroblasts present in different skin conditions (n=4 mice per condition; ** p<0.01; *** p<0.001; **** p<0.0001; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M. c. Herovici staining of sections of normal (WT), inflamed (InvEE), wound, or tumour-associated (Papilloma) skin. Scale bars: 100 μm.


Figure 3c . Relative mRNA levels of PRSS35 in lysates from normal skin ( WT , n = 4 mice ) , inflamed skin ( InvEE , n = 4 mice ) , wounds ( d14pw , n = 4 mice ) and wound - induced papillomas ( Pap , n = 5 mice ) . ( * p = 0 . 0159 ; Mann - Whitney test ) . Data represent means of three technical replicates ± S . E . M . f . H & E - stained skin sections of WT and PRSS35 - / - wounds at different days post - wounding . Scale bars : 100 µm .

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Figure 3c. Relative mRNA levels of PRSS35 in lysates from normal skin (WT, n=4 mice), inflamed skin (InvEE, n=4 mice), wounds (d14pw, n=4 mice) and wound-induced papillomas (Pap, n=5 mice). (* p=0.0159; Mann-Whitney test). Data represent means of three technical replicates ± S.E.M.f. H&E-stained skin sections of WT and PRSS35-/- wounds at different days post-wounding. Scale bars: 100 µm.


Figure 4c , d . Incidence of papilloma formation ( ns : non - significant ; Gehan - Breslow - Wilcoxon test ) ( c ) and number of tumours per mouse ( d ) in wild - type ( WT , n = 17 ) and PRSS35 - / - ( n = 22 ) mice treated with DMBA - TPA ( * p = 0 . 0171 ; Wilcoxon matched - paired rank test ) . Data represent means ± S . E . M .

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Figure 4c, d. Incidence of papilloma formation (ns: non-significant; Gehan-Breslow-Wilcoxon test) (c) and number of tumours per mouse (d) in wild-type (WT, n=17) and PRSS35-/- (n=22) mice treated with DMBA-TPA (*p=0.0171; Wilcoxon matched-paired rank test). Data represent means ± S.E.M.


Figure 5b . Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures ( col1a1 : collagen type 1 alpha 1 ; αSMA , alpha smooth muscle actin ; Cyr61 : Cysteine - rich angiogenic inducer 61 ( = CCN1 ) ) in primary fibroblast cultures isolated from WT and PRSS35 - / - mice , untreated or after 24 hours stimulation with 10 ng / mL TGFβ1 ( n ≥ 7 biological replicates per condition ) ( * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M . c Relative mRNA expression levels of the indicated cytokines / growth factors in primary fibroblast cultures ( n ≥ 8 per condition ) ( VEGF - α , vascular endothelial growth factor - alpha ; FGF2 : basic fibroblast growth factor ; FGF7 : keratinocyte growth factor ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M . d . Picrosirius red staining of normal skin , day 14 post - wounding ( d14pw ) skin or wound - induced papillomas from WT and PRSS35 KO mice . Red colouring indicates thick fibres , green colouring indicates thin fibres . Scale bars : 200 µm . e . Quantification of picrosirius red staining as total collagen density ( upper panel ) and ratio of red versus green picrosirius staining ( lower panel ) . Using QuPath Bioimage analysis software pixels colouring red or green were determined per area . ( n ≥ 4 technical replicates per condition ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M .

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Figure 5b. Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures (col1a1: collagen type 1 alpha 1; αSMA, alpha smooth muscle actin; Cyr61: Cysteine-rich angiogenic inducer 61 (=CCN1)) in primary fibroblast cultures isolated from WT and PRSS35-/- mice, untreated or after 24 hours stimulation with 10 ng/mL TGFβ1 (n≥7 biological replicates per condition) (** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M. c Relative mRNA expression levels of the indicated cytokines/growth factors in primary fibroblast cultures (n≥8 per condition) (VEGF-α, vascular endothelial growth factor-alpha; FGF2: basic fibroblast growth factor; FGF7: keratinocyte growth factor) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M. d. Picrosirius red staining of normal skin, day 14 post-wounding (d14pw) skin or wound-induced papillomas from WT and PRSS35 KO mice. Red colouring indicates thick fibres, green colouring indicates thin fibres. Scale bars: 200 µm. e. Quantification of picrosirius red staining as total collagen density (upper panel) and ratio of red versus green picrosirius staining (lower panel). Using QuPath Bioimage analysis software pixels colouring red or green were determined per area. (n≥4 technical replicates per condition) (* p<0.05, ** p<0.01, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.


Figure 6a . Human skin sections were stained with a PRSS35 - specific antibody ( red ) and counterstained with Dapi ( n = 5 per condition ) . Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal - dermal boundary . Scale bars : 100 μm . b . Quantification of PRSS35 positive signal by Volocity analysis ( n ≥ 4 per condition ; * * p < 0 . 01 ; One - way ANOVA testing ) . Data represent means of multiple microscopic fields ± S . E . M .

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Figure 6a. Human skin sections were stained with a PRSS35-specific antibody (red) and counterstained with Dapi (n=5 per condition). Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal-dermal boundary. Scale bars: 100 μm. b. Quantification of PRSS35 positive signal by Volocity analysis (n≥4 per condition; ** p<0.01; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M.


Figure 3 ( C ) Cleavage of supercoiled ( SC ) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants . Efficient full length linearization ( FLL ) of the substrate resulting from a complete double - strand break was only observed when using sgRNAs bearing the 5 ' most end of the tracrRNA ( variants 1 and 3 ) . Moreover , the terminator - like Hairpin 3 is not required for target cleavage ( variant 1 ) . OC - open circular ; FLL - full length linear ; SC - supercoiled .

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Figure 3(C) Cleavage of supercoiled (SC) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.


Figure 4 ( B ) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l . wt - wildtype . ( C ) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition . The molar ratio of RNP to substrate was kept low ( 5 : 1 ) to increase reaction stringency . Asp2Cas12l predominantly recognizes a 5 ' - CCY - 3 ' PAM and Asp3Cas12l a 5 ' - CCB - 3 ' PAM . N = 3 . Data information : In ( C ) , individual data points are plotted , where n = 4 replicates from independent experiments ( Asp2Cas12l ) and n = 3 replicates from independent experiments ( Asp3Cas12l ) . The data points were fitted to a single exponential association curve ( solid lines ) .

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Figure 4(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3. Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).


Figure 5 ( A ) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single - stranded ( ss ) DNA or dsDNA ( including PAM ) activators with target sequences complementary to the gRNA spacer . NS activator - non specific activator ( ssDNA oligonucleotide or dsDNA duplex ) with no sequence complementarity to the spacer of the gRNA . ( B ) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes . Background - subtracted traces from fluorescent reporter assays with favored ssDNA ( 5 ' - CCCCCCCC - 3 ' ) or ssRNA ( 5 ' - CCCCCCCC - 3 ' ) probes . RFU - relative fluorescence units . ( C ) Background - subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes . ( D ) Rates of trans - degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets . Collateral ssDNAse activity is about 3 - fold higher than the rate of ssRNA degradation . Fluorescence intensities in B - C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters . Data information : In ( B - D ) , individual data points from n = 3 replicates from independent experiments are plotted . In ( D ) , bars represent the mean .

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Figure 5(A) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single-stranded (ss) DNA or dsDNA (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.(B) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA (5'-CCCCCCCC-3') or ssRNA (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units. (C) Background-subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes. (D) Rates of trans-degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation. Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters. Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.