Figure 5PI ( 5 ) P Triggers Autophagy in the Absence of VPS34 Activation ( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells pretreated with Wm ( 200 nM , 2 hr ) , then loaded with 10 μM PI ( 5 ) P for 1 hr ( in the presence of Wm and BAF ) ( mean ± SEM ) . ( C ) Quantification of numbers of GFP - LC3 vesicles in HeLa cells stably expressing GFP - LC3 treated as in ( A ) and then shifted to starvation media ( HBSS ) or complete media ( Basal ) for 2 hr ( in the presence of Wm ) ( mean ± SEM ) . ( D ) HeLa cells transfected with HA - ATG12 and ATG5 treated with 200 nM Wm as in ( A ) and subjected to western blot analysis with anti - HA antibody to detect free ATG12 and the ATG5 - ATG12 complex ( mean ± SEM ) . ATG5 - ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti - ATG12 antibody ( bottom ) . ( E and F ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells transfected with control , PI5P4K2A , 2B , and 2C siRNA and treated with Wm ( 200 nM , 2 hr in the presence of BAF ) ( mean ± SEM ) . ( G and H ) Western blot analysis of free ATG12 and ATG5 - ATG12 complex levels with anti - HA antibody in HeLa cells treated with control , PI5P4K2A , 2B , and 2C siRNA , transfected with HA - ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr ( mean ± SEM ) . ( I ) HeLa cells stably expressing GFP - LC3 treated with control , PI5P4K2A , 2B , and 2C siRNA , were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr ( in the presence of Wm ) ( mean ± SEM ) . ( J and K ) HeLa cells stably expressing GFP - LC3 were transfected for 30 hr with myc - tagged empty vector , myc - MTMR3WT , and myc - MTMR3C413S , incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm . Cells were fixed , stained with anti - myc antibodies , and imaged by confocal microscope . Asterisks indicate transfected cells . Quantification of numbers of GFP - LC3vesicles per cell is shown in ( K ) ( mean ± SEM ) . ( J and K ) HeLa cells stably expressing GFP - LC3 were transfected for 30 hr with myc - tagged empty vector , myc - MTMR3WT , and myc - MTMR3C413S , incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm . Cells were fixed , stained with anti - myc antibodies , and imaged by confocal microscope . Asterisks indicate transfected cells . Quantification of numbers of GFP - LC3 vesicles per cell is shown in ( K ) ( mean ± SEM ) . ( L and M ) HeLa cells transfected with GFP - PI5P4K2A , 2B , 2C , and RFP - LC3 for 30 hr were loaded with 10 μM PI ( 3 ) P for 1 hr in complete medium , and then fixed and imaged on confocal microscope . Bar , 10 μm . Quantification of cells ( percentage of total ) showing more than 10 RFP - LC3 vesicles in the different conditions from ( J ) is shown in ( M ) ; n = 200 cells ( mean ± SEM ) . See also Figure S5 .

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

Figure 5PI(5)P Triggers Autophagy in the Absence of VPS34 Activation(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells pretreated with Wm (200 nM, 2 hr), then loaded with 10 μM PI(5)P for 1 hr (in the presence of Wm and BAF) (mean ± SEM).(C) Quantification of numbers of GFP-LC3 vesicles in HeLa cells stably expressing GFP-LC3 treated as in (A) and then shifted to starvation media (HBSS) or complete media (Basal) for 2 hr (in the presence of Wm) (mean ± SEM).(D) HeLa cells transfected with HA-ATG12 and ATG5 treated with 200 nM Wm as in (A) and subjected to western blot analysis with anti-HA antibody to detect free ATG12 and the ATG5-ATG12 complex (mean ± SEM). ATG5-ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti-ATG12 antibody (bottom).(E and F) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected with control, PI5P4K2A, 2B, and 2C siRNA and treated with Wm (200 nM, 2 hr in the presence of BAF) (mean ± SEM).(G and H) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA, transfected with HA-ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr (mean ± SEM).(I) HeLa cells stably expressing GFP-LC3 treated with control, PI5P4K2A, 2B, and 2C siRNA, were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr (in the presence of Wm) (mean ± SEM).(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3vesicles per cell is shown in (K) (mean ± SEM).(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3 vesicles per cell is shown in (K) (mean ± SEM).(L and M) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, and RFP-LC3 for 30 hr were loaded with 10 μM PI(3)P for 1 hr in complete medium, and then fixed and imaged on confocal microscope. Bar, 10 μm. Quantification of cells (percentage of total) showing more than 10 RFP-LC3 vesicles in the different conditions from (J) is shown in (M); n = 200 cells (mean ± SEM). See also Figure S5.


Figure 6 ( A ) HeLa cells transfected with GFP - WIPI2B or GFP - DFCP1 preloaded with indicated concentrations of PI ( 5 ) P for 1 hr , starved in HBSS for 1 hr , and then incubated with Wm in HBSS were tracked by time - lapse microscopy for 10 min after the addition of Wm . Quantification of WIPI2B or DFCP1vesicles ( percentage of those at the starting time [ T0 ] ) during the treatments are shown in the graphs . ( B - D ) HeLa cells treated with control , PI5P4K2A , 2B , and 2C siRNA were transfected with GFP - WIPI2B ( B ) , GFP - WIPI2D ( C ) , or GFP - DFCP1 ( D ) ; starved in HBSS ( 1 hr ) ; and then incubated with Wm in HBSS . WIPI2 or DFCP1 structures were tracked and quantified as in ( A ) . ( E ) Lysates from HeLa cells stably expressing GFP - WIPI2B were incubated with agarose beads coated with PI , PI ( 5 ) P , and PI ( 3 ) P , eluted with SDS - PAGE sample buffer , and recovered proteins were assessed by western blotting using antibodies against GFP . Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls . ( F - H ) Cell extracts from HeLa cells stably expressing GFP - WIPI2B were incubated for 3 hr with PI ( 5 ) P - containing liposomes ( F and H ) or PI ( 3 ) P - containing liposomes ( G and H ) before a pull - down experiment using the indicated beads . PS - containing liposomes were used as internal controls for both competition assays in ( F and G ) . ( I ) Lysates from HeLa cells stably expressing GFP - WIPI2B WT and GFP - WIPI2B FTTG mutant were incubated with agarose beads coated with PI ( 5 ) P and PI ( 3 ) P , eluted with SDS - PAGE sample buffer , and recovered proteins were assessed by western blotting using antibodies against GFP . See also Figure S6 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(A) HeLa cells transfected with GFP-WIPI2B or GFP-DFCP1 preloaded with indicated concentrations of PI(5)P for 1 hr, starved in HBSS for 1 hr, and then incubated with Wm in HBSS were tracked by time-lapse microscopy for 10 min after the addition of Wm. Quantification of WIPI2B or DFCP1vesicles (percentage of those at the starting time [T0]) during the treatments are shown in the graphs.(B-D) HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA were transfected with GFP-WIPI2B (B), GFP-WIPI2D (C), or GFP-DFCP1 (D); starved in HBSS (1 hr); and then incubated with Wm in HBSS. WIPI2 or DFCP1 structures were tracked and quantified as in (A).(E) Lysates from HeLa cells stably expressing GFP-WIPI2B were incubated with agarose beads coated with PI, PI(5)P, and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls.(F-H) Cell extracts from HeLa cells stably expressing GFP-WIPI2B were incubated for 3 hr with PI(5)P-containing liposomes (F and H) or PI(3)P-containing liposomes (G and H) before a pull-down experiment using the indicated beads. PS-containing liposomes were used as internal controls for both competition assays in (F and G).(I) Lysates from HeLa cells stably expressing GFP-WIPI2B WT and GFP-WIPI2B FTTG mutant were incubated with agarose beads coated with PI(5)P and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. See also Figure S6.


Figure 7 ( A ) HeLa cells stably expressing GFP - LC3 were shifted to starvation media ( HBSS or glucose - free DMEM ) for 4 hr ( in the presence of 200 nM Wm or 100 nM YM - 201636 ) . ( B ) Quantification of numbers of GFP - LC3 vesicles per cell in HeLa cells stably expressing GFP - LC3 treated as in ( A ) is shown in the graph ( mean ± SEM ) . ( C ) Western blot analysis of LC3 - II and tubulin levels in MEF and SKNSH cells were treated as in ( A ) in complete media or glucose - free media in the presence of 100 nM YM - 201636 , 200 nM Wm , and 400 nM BAF for 4 hr . ( D ) HeLa cells transfected with GFP - PHD3X and Strawberry - ATG16L1 for 16 hr were left in complete media ( basal ) or glucose - free media ( glucose free ) for 4 hr in the presence or absence of 100 nM YM - 201636 , then fixed and imaged on a confocal microscope . ( E and F ) HeLa cells transfected with GFP - PI5P4K2A , 2B , 2C , or PI5P4K2C catalytic dead and RFP - LC3 for 30 hr ( E ) were starved for glucose for 4 hr , then fixed and imaged on a confocal microscope . Bar , 10 μm . ( F ) Quantification of cells ( percentage of total ) showing more than ten autophagic vesicles ( RFP - LC3 vesicles ) in the different conditions from ( F ) is shown in the graph ; n = 200 cells ( mean ± SEM ) . ( G ) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP - PHD3X , Strb - ATG16L1 , and myc - MTMR3 WT . Cells were starved for glucose for 4 hr , then fixed , stained with anti - myc antibodies , and imaged on a confocal microscope . Bar , 10 μm . ( H ) HeLa cells transfected for 16 hr with GFP - PHD3X and Strb - ATG16L1 were starved for 4 hr in HBSS or glucose - free media , then fixed , stained for PI ( 3 ) P antibodies , and imaged on a confocal microscope . Bar , 10 μm . See also Figure S7 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7(A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).(B) Quantification of numbers of GFP-LC3 vesicles per cell in HeLa cells stably expressing GFP-LC3 treated as in (A) is shown in the graph (mean ± SEM).(C) Western blot analysis of LC3-II and tubulin levels in MEF and SKNSH cells were treated as in (A) in complete media or glucose-free media in the presence of 100 nM YM-201636, 200 nM Wm, and 400 nM BAF for 4 hr.(D) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in complete media (basal) or glucose-free media (glucose free) for 4 hr in the presence or absence of 100 nM YM-201636, then fixed and imaged on a confocal microscope.(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or PI5P4K2C catalytic dead and RFP-LC3 for 30 hr (E) were starved for glucose for 4 hr, then fixed and imaged on a confocal microscope. Bar, 10 μm. (F) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM).(G) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP-PHD3X, Strb-ATG16L1, and myc-MTMR3 WT. Cells were starved for glucose for 4 hr, then fixed, stained with anti-myc antibodies, and imaged on a confocal microscope. Bar, 10 μm.(H) HeLa cells transfected for 16 hr with GFP-PHD3X and Strb-ATG16L1 were starved for 4 hr in HBSS or glucose-free media, then fixed, stained for PI(3)P antibodies, and imaged on a confocal microscope. Bar, 10 μm. See also Figure S7.


Figure 1 . ( A ) Flow cytometry analysis of l ( 2 ) mbn cells starved for 2 h ( 2h S ) or 4 h ( 4h S ) showed an increase in LTG fluorescence levels ( x axis ) compared with control cells in full - nutrient medium ( C ) . The gate shown on the histogram represents the LTGhigh population . ( B ) Representative images of GFP - LC3 puncta in control and 2 - h starved l ( 2 ) mbn cells . An increase in GFP - LC3 puncta was observed in the starved cells ( left ) . Bar , 10 μm . ( C ) Flow cytometry analysis of 4 - h starved cells were incubated with 3MA ( 4h S + 3MA ) and Baf ( 4h S + Baf ) . Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells ( 4h S ) . Control cells in nutrient - full medium ( C ) are represented by the brown line . ( D ) Both autophagy inhibitors , 3MA and Baf , reduced the LTGhigh population significantly . ( 3MA , P = 0 . 00001 ; and Baf , P = 0 . 00006 ) . ( E ) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control . ( Atg1 , P = 0 . 01 ; Atg5 , P = 0 . 01 ; Atg7 , P = 0 . 002 ; Atg8a , P = 0 . 008 ; Atg8b , P = 0 . 006 ; and Atg12 , P = 0 . 02 ) . ( F ) RNAi of all tested genes in the TOR - PI3K pathways had a statistically significant effect on LTG fluorescence levels . Known negative regulators of autophagy are shown with gray bars ; positive regulators are shown with white bars . ( Pten , P = 0 . 007 ; Tsc1 , P = 0 . 027 ; Tsc2 , P = 0 . 025 ; RheB , P = 0 . 005 ; Tor , P = 0 . 016 ; and S6k , P = 0 . 012 ) . dsRNA corresponding to a human gene ( Hs ) was used as a negative control in E and F . Results represent the mean value ± SD from at least three independent experiments .

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

Figure 1.(A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006).(E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02).(F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.


Figure 2 . ( A ) The percentage of LTGhigh cells was reduced by hid - RNAi ( P = 0 . 006 ) but not by rpr , grim , and skl - RNAi . ( B ) Knockdown of Ras , phl , and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells . ( Ras , P = 0 . 003 ; phl , P = 0 . 001 ; and rl , P = 0 . 028 ) . ( C ) th - RNAi treatment ( 24 h ) had no significant effect on LTG levels ; in contrast , RNAi of Bruce resulted in an increase in LTG fluorescence levels ( P = 0 . 01 ) . ( D ) Reduction of debcl , Buffy , or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels . ( debcl , P = 0 . 018 ; Buffy , P = 0 . 006 ; and p53 , P = 0 . 004 ) . ( E ) RNAi of effector caspase Dcp - 1 resulted in a significant decrease in the LTGhigh population ( P = 0 . 001 ) . ( F ) Representative images of GFP - LC3 puncta in cells treated with the indicated RNAi after a 2 - h starvation treatment . Bar , 10 μm . ( G ) Quantification of cells with GFP - LC3 puncta after RNAi treatment . Cells with more than three GFP - LC3 punctate dots were considered to be GFP - LC3 - positive cells . Cells treated with the RNAi indicated here all showed a significant difference ( P < 0 . 05 ) in the percentage of GFP - LC3 - positive cells compared with the human ( Hs ) RNAi control . ( Pten , P = 0 . 006 ; Tor , P = 0 . 034 ; Buffy , P = 0 . 005 ; debcl , P = 0 . 003 ; Bruce , P = 0 . 003 ; Dcp - 1 , P = 0 . 007 ; hid , P = 0 . 002 ; Ras , P = 0 . 006 ; and phl , P = 0 . 050 ) . Results represent the mean value ± SD from three independent experiments .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi.(B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028).(C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01).(D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004).(E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001).(F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm.(G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3-positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3-positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.


Figure 3 . ( A ) GFP - LC3 proteins were expressed in nurse cells ( NC ) but not in follicle cells ( FC ) by using the UASp / nanos Gal4 system . DAPI staining of nuclei is shown in blue . ( B ) UASp - GFP - LC3 ; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP - LC3 pattern . Numerous GFP - LC3 puncta ( green ) at region 2 within germarium were observed in nutrient - deprived flies . Ovaries were stained with LTR in w1118 flies . Germarium of nutrient - deprived w1118 flies had an increase in punctate LTR staining ( red ) compared with well - fed germarium . ( C ) Degenerating stage 8 egg chambers ( arrows ) had numerous GFP - LC3 puncta ( green ) and an increase in LTR - positive dots ( red ) compared with healthy egg chambers ( arrowheads ) . DAPI ( blue ) staining of nuclei is shown in the two panels on the right . ( D ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) showed a dramatic decrease in LTR staining . DAPI staining of nuclei is shown in blue .

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

Figure 3.(A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue.(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.(C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right.(D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue.


Figure 4 . ( A ) Germaria of the nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR staining compared with nutrient - deprived wild - type flies shown in Fig . 3 B . DAPI staining of nuclei is shown in blue . ( B ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR staining compared with nutrient - deprived wild - type flies shown in Fig . 3 C . ( C ) Lack of Dcp - 1 function ( UASp - GFP - LC3 Dcp - 1Prev / Dcp - 1Prev ; nanos - GAL4 / + ) resulted in uniform diffuse staining of GFP - LC3 rather than the punctate pattern observed in wild - type degenerating stage 8 egg chambers shown in Fig 3 C . ( D ) Dying egg chambers ( arrows ) of NGT / + ; nanos - GAL4 / UASp - fl - Dcp - 1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining ( red ) compared with healthy egg chambers ( arrowheads ) . ( E ) Expression of activated Dcp - 1 ( a truncated form ) and GFP - LC3 in the germ line ( UASp - GFP - LC3 / + ; nanos - GAL4 / nanos - GAL4 UASp - tDcp - 1 ) resulted in abundant degenerating stage 8 egg chambers ( arrows ) with numerous GFP - LC3 puncta ( green ) . DAPI staining of nuclei is shown in blue .

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

Figure 4.(A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C.(C) Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1Prev/Dcp-1Prev; nanos-GAL4/+) resulted in uniform diffuse staining of GFP-LC3 rather than the punctate pattern observed in wild-type degenerating stage 8 egg chambers shown in Fig 3 C. (D) Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared with healthy egg chambers (arrowheads). (E) Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germ line (UASp-GFP-LC3/+; nanos-GAL4/nanos-GAL4 UASp-tDcp-1) resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in blue.


Figure 5 . ( A ) The germarium in well - fed BruceE81 flies showed an increase in LTR staining ( red ) compared with wild - type well - fed flies shown in Fig . 3 B ( top right ) . DAPI staining ( white ) of nuclei is shown on the right . ( B ) In well - fed wild - type flies , mid - oogenesis nurse cell death is a rare event . Lack of Bruce function resulted in an increase in dying stage 8 egg chambers ( arrows ) in ovaries under well - fed conditions , and these degenerating stage 8 egg chambers had numerous LTR ( red ) punctate dots . DAPI staining ( white ) is shown on the right . Bars : ( A ) 20 μm ; ( B ) 50 μm .

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

Figure 5.(A) The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared with wild-type well-fed flies shown in Fig. 3 B (top right). DAPI staining (white) of nuclei is shown on the right. (B) In well-fed wild-type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well-fed conditions, and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.


Figure 6 . ( A ) Ovaries were stained with TUNEL ( green ) to detect DNA fragmentation . Clusters of cysts with TUNEL staining were observed in region 2 in nutrient - deprived w1118 files . In Dcp - 1Prev flies , fewer TUNEL - positive cysts in region 2 were observed . Under well - fed conditions , numerous TUNEL - positive cysts were observed in BruceE81 flies . DAPI staining of nuclei is shown in white . ( B ) Numerous degenerating stage 8 egg chambers ( arrows ) with Bruce - positive staining ( green ) were observed in well - fed BruceE81 flies . DAPI staining of nuclei ( white ) is shown on the right .

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

Figure 6.(A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with Bruce-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right.


Figure 7 . ( A ) TUNEL - positive staining was observed in dying stage 8 egg chambers ( arrows ) of starved control flies ( CG5335d30 / Atg7d14 ) . DAPI staining of nuclei ( white ) is shown on the right . ( B ) In nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) , degenerating stage 8 egg chambers ( arrows ) showed no or low levels of TUNEL staining . Nuclear DNA condensation , detected by DAPI , was still observed . ( C ) Dying stage 8 egg chambers ( arrows ) from nutrient - deprived control siblings ( Atg1Δ3D / TM3 ) generated from the same cross in D had abundant TUNEL - positive staining . ( D ) In nutrient - deprived Atg1 GLCs , degenerating stage 8 egg chambers ( arrows ) showed no or low levels of TUNEL staining . Nuclear DNA condensation ( DAPI , right ) in degenerating egg chambers appeared to occur as in the controls .

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

Figure 7.(A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls.


Figure 2B : U2 - OS cells were transfected with the indicated siRNAs . The indicated cells were treated with etoposide ( 50 µM , 90 min . before analysis ) . Whole - cell extracts were immunoblotted with the indicated antibodies . The result is representative of three biological replicates . D : U2 - OS cells were transfected with non - targeting ( mock ) or TSG101 - targeting siRNAs and irradiated ( 20 Gy ) 90 min . before analysis . Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one - way ANOVA ( * p < 0 . 05 ) in the right panel .

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Figure 2B: U2-OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 µM, 90 min. before analysis). Whole-cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.D: U2-OS cells were transfected with non-targeting (mock) or TSG101-targeting siRNAs and irradiated (20 Gy) 90 min. before analysis. Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one-way ANOVA (*p < 0.05) in the right panel.


Figure 3A : Nuclear - cytoplasmic fractionation of U2 - OS cells . Indicated samples were transfected with siRNAs against TSG101 . PARylation was inhibited by olaparib ( 10 µM , 24 hours before analysis ) . Cytoplasmic export of p - ATM was induced by etoposide ( 50 µM , 45 minutes before analysis ) . Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies . The fractionation efficiency was controlled by the respective subcellular marker proteins ( PARP1 and LDH - A ) . PARylation was detected using the Pan ADPr reagent . Note that PARylation is activated by shearing forces during extract preparation ( lane 1 ) B : U2 - OS cells were transfected with non - targeting ( - ) or TSG101 - targeting ( + ) siRNAs and cells were irradiated ( + ) or not ( - ) as indicated ( 20 Gy ) 10 min . before analysis . Whole - cell extracts were immunoblotted with the 10H PAR monoclonal antibody . C : siRNA - transfected U2 - OS cells as in B were etoposide - treated or not , as indicated . Whole - cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly - and mono - ( ADP - ribose ) . Note that PARylation also occurred during cell lysis in untreated cells . D : U2 - OS cells were transfected with non - targeting or VPS28 - targeting siRNAs . Whole - cell extracts were immunoblotted with indicated antibodies . F : Cells were irradiated ( 20 Gy ) as indicated 10 min before analysis . Whole - cell extracts were immunoblotted with the 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O 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: Nuclear-cytoplasmic fractionation of U2-OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 µM, 24 hours before analysis). Cytoplasmic export of p-ATM was induced by etoposide (50 µM, 45 minutes before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH-A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1)B: U2-OS cells were transfected with non-targeting (-) or TSG101-targeting (+) siRNAs and cells were irradiated (+) or not (-) as indicated (20 Gy) 10 min. before analysis. Whole-cell extracts were immunoblotted with the 10H PAR monoclonal antibody.C: siRNA-transfected U2-OS cells as in B were etoposide-treated or not, as indicated. Whole-cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly- and mono-(ADP-ribose). Note that PARylation also occurred during cell lysis in untreated cells.D: U2-OS cells were transfected with non-targeting or VPS28-targeting siRNAs. Whole-cell extracts were immunoblotted with indicated antibodies.F: Cells were irradiated (20 Gy) as indicated 10 min before analysis. Whole-cell extracts were immunoblotted with the indicated antibodies.


Figure 4A : Proximity ligation assay ( PLA ) assay with unstimulated , etoposide ( Eto ) - treated ( 50 µM , 10 min . before analysis ) , or etoposide plus olaparib - ( Olap ) co - treated ( 50 µM of etoposide , 10 min . before analysis and 10 µM of olaparib 24 hours before analysis ) U2 - OS cells . The red dots represent PARP1 - TSG101 interaction . The nucleus is stained with DAPI ( blue ) . Scale bar : 10 µm . As negative controls , PLA was performed without primary PARP1 or TSG101 antibodies . As an additional control , PLA was performed in PARP1 depleted cells to show specificity B : Quantification of PLA dots per nuclei in A . Blind counting was performed from 30 replicates three biologically independent experiments . The conditions were compared with an ordinary one - way ANOVA ( ns , p > 0 . 05 ; * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . C : U2 - OS cells were cotransfected with the indicated deletion constructs of mCherry - tagged TSG101 and EGFP - tagged PARP1 . Immunoprecipitation of GFP was performed using whole - cell extracts . F : In - vitro PARylation reactions were carried out with the indicated recombinant and purified proteins , including 8 ng PARP1 in the presence of sssDNA ( sheared salmon sperm DNA ) and MgCl2 . 100 ng of TSG101 was added to the reaction , as indicated . The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls .

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

Figure 4A: Proximity ligation assay (PLA) assay with unstimulated, etoposide (Eto)-treated (50 µM, 10 min. before analysis), or etoposide plus olaparib-(Olap) co-treated (50 µM of etoposide, 10 min. before analysis and 10 µM of olaparib 24 hours before analysis) U2-OS cells. The red dots represent PARP1-TSG101 interaction. The nucleus is stained with DAPI (blue). Scale bar: 10 µm. As negative controls, PLA was performed without primary PARP1 or TSG101 antibodies. As an additional control, PLA was performed in PARP1 depleted cells to show specificity B: Quantification of PLA dots per nuclei in A. Blind counting was performed from 30 replicates three biologically independent experiments. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001).C: U2-OS cells were cotransfected with the indicated deletion constructs of mCherry-tagged TSG101 and EGFP-tagged PARP1. Immunoprecipitation of GFP was performed using whole-cell extracts.F: In-vitro PARylation reactions were carried out with the indicated recombinant and purified proteins, including 8 ng PARP1 in the presence of sssDNA (sheared salmon sperm DNA) and MgCl2. 100 ng of TSG101 was added to the reaction, as indicated. The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls.


Figure 5B : Representative images of PARP1 - GFP association with laser - microirradiation sites in untreated ( mock ) , olaparib - treated ( 10 µM , 24 hours before analysis ) or siTSG101 - transfected U2 - OS cells at indicated times post - irradiation . Scale bar : 7 . 5 µm . The image is representative of nine replicates from three biologically independent experiments . Go to Movies EV1 - 3 for further illustration . C : Kinetics of PARP1 - GFP recruitment and dissociation from DNA lesions were measured at times indicated . Nine nuclei were analyzed for each indicated condition from three biologically independent experiments . The data are shown as mean GFP intensity in the microirradiated area ± SEM , normalized to the mean GFP intensity of corresponding whole nuclei . D : Relative PARP1 - GFP recruitment to DNA lesions for each indicated condition at 12 min . post - irradiation .

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

Figure 5B: Representative images of PARP1-GFP association with laser-microirradiation sites in untreated (mock), olaparib-treated (10 µM, 24 hours before analysis) or siTSG101-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 7.5 µm. The image is representative of nine replicates from three biologically independent experiments. Go to Movies EV1-3 for further illustration.C: Kinetics of PARP1-GFP recruitment and dissociation from DNA lesions were measured at times indicated. Nine nuclei were analyzed for each indicated condition from three biologically independent experiments. The data are shown as mean GFP intensity in the microirradiated area ± SEM, normalized to the mean GFP intensity of corresponding whole nuclei. D: Relative PARP1-GFP recruitment to DNA lesions for each indicated condition at 12 min. post-irradiation.


Figure 6C : Cells were transfected with non - targeting or TSG101 - targeting siRNAs . Immunofluorescence staining was performed with the indicated antibodies ( red : cleaved caspase 3 ; green : γH2AX ) . Nuclei were stained with DAPI ( blue ) . The image is a representative of 3 biologically independent experiments . Scale bar : 30 µm . See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR - treatment . D : Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments . Conditions were compared with an unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 ) . E : γH2AX staining of TSG101 - targeting or non - targeting siRNA transfected U2 - OS cells reveals puncta - like foci at 2 or 7 days after irradiation . F : The number of puncta - like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments .

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Figure 6C: Cells were transfected with non-targeting or TSG101-targeting siRNAs. Immunofluorescence staining was performed with the indicated antibodies (red: cleaved caspase 3; green: γH2AX). Nuclei were stained with DAPI (blue). The image is a representative of 3 biologically independent experiments. Scale bar: 30 µm. See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR-treatment. D: Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (*p < 0.05, **p < 0.01).E: γH2AX staining of TSG101-targeting or non-targeting siRNA transfected U2-OS cells reveals puncta-like foci at 2 or 7 days after irradiation.F: The number of puncta-like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments.


Figure 1 ( A ) In situ hybridizations for Elp3 transcripts in the developing inner ear . Transcripts are present at E12 . 5 in a ventro - medial region of the developing cochlear duct ( CD ) and in the cochleo - vestibular ganglion ( CVG ) . At E16 . 5 , transcripts are present in the spiral ganglion neurons ( SGNs ) and in the newly formed hair cells ( HCs ) at the base of the cochlea , where they remain strongly expressed at P1 . Scale bar = 50µm ; inset = 25µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 , OC : Organ of Corti . ( B ) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth . Scale bar = 10µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 , OC : Organ of Corti . Validation of Elp3 depletion from the cochlea in Elp3cKO animals . ( C ) At P1 , Elp3 mRNA is absent from Elp3cKO . Scale bars = 50µm . In situ hybridizations were repeated 4 times . Validation of Elp3 depletion from the cochlea in Elp3cKO animals . ( D ) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level ( cochleae from 3 different animals are pooled per sample ) . ( E ) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound - evoked potentials in Elp3cKO animals , indicating that they suffer from severe hearing loss . ( F ) Elp3cKO animals present increased ABR thresholds ( WT / Elp3cKO n = 5 / 3 . Unpaired two - tailed t - test , * * p = 0 . 0017 , t = 5 . 369 , DF = 6 , mean ± SD ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In situ hybridizations for Elp3 transcripts in the developing inner ear. Transcripts are present at E12.5 in a ventro-medial region of the developing cochlear duct (CD) and in the cochleo-vestibular ganglion (CVG). At E16.5, transcripts are present in the spiral ganglion neurons (SGNs) and in the newly formed hair cells (HCs) at the base of the cochlea, where they remain strongly expressed at P1. Scale bar=50µm; inset=25µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.(B) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth. Scale bar=10µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (C) At P1, Elp3 mRNA is absent from Elp3cKO. Scale bars=50µm. In situ hybridizations were repeated 4 times.Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (D) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level (cochleae from 3 different animals are pooled per sample).(E) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound-evoked potentials in Elp3cKO animals, indicating that they suffer from severe hearing loss. (F) Elp3cKO animals present increased ABR thresholds (WT/Elp3cKO n=5/3. Unpaired two-tailed t-test, **p=0.0017, t=5.369, DF=6, mean±SD).


Figure 2 ( A ) Toluidine blue stainings of semi - thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss . Scale bar = 100µm , OC : Organ of Corti . ( B ) Specific labellings of the pre - synaptic marker Ctbp2 ( in IHCs ) and the post - synaptic marker GluR2 / 3 ( in SGN terminals ) indicate a decrease in the number of double - labelled dots in P15 Elp3cKO whole - mounted cochlea compared to WT . The remaining synapses still show coupling of the pre - and post - synaptic markers , suggesting they may be functional . Scale bar = 20µm . ( C ) The mean number of Ctbp2 - GluR2 / 3 double - positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65 % compared to control ( n = 6 animals per genotype ; Unpaired two - tailed t - test , * * * p < 0 . 001 , t = 13 . 48 , DF = 10 , mean ± SD ) . ( D ) NF - 1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae . Scale bar = 100µm . ( E ) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14 . 5 onwards ( n = 3 - 6 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 ; * p < 0 . 05 ; F = 20 . 43 ; DF = 7 ; mean ± SD ) . ( F ) Detection of apoptotic SGNs from E13 . 5 cochlear sections with cleaved caspase - 3 and B - tubIII stainings . Scale bar = 100µm . ( G ) The number of apoptotic SGN per section is significantly increased in E13 . 5 and E14 . 5 Elp3cKO compared to WT cochleae , suggesting SGNs are properly specified but die during their differentiation process ( n = 3 - 4 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 ; * * p < 0 . 01 ; F = 23 . 74 ; DF = 7 ; mean ± SD ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar=100µm, OC: Organ of Corti.(B) Specific labellings of the pre-synaptic marker Ctbp2 (in IHCs) and the post-synaptic marker GluR2/3 (in SGN terminals) indicate a decrease in the number of double-labelled dots in P15 Elp3cKO whole-mounted cochlea compared to WT. The remaining synapses still show coupling of the pre- and post-synaptic markers, suggesting they may be functional. Scale bar=20µm. (C) The mean number of Ctbp2-GluR2/3 double-positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65% compared to control (n=6 animals per genotype; Unpaired two-tailed t-test, ***p<0.001, t=13.48, DF=10, mean±SD). (D) NF-1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae. Scale bar=100µm.(E) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14.5 onwards (n=3-6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; *p<0.05; F=20.43; DF=7; mean±SD).(F) Detection of apoptotic SGNs from E13.5 cochlear sections with cleaved caspase-3 and B-tubIII stainings. Scale bar=100µm.(G) The number of apoptotic SGN per section is significantly increased in E13.5 and E14.5 Elp3cKO compared to WT cochleae, suggesting SGNs are properly specified but die during their differentiation process (n=3-4 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; **p<0.01; F=23.74; DF=7; mean±SD).


Figure 3 ( A ) Proteostat staining of WT and Elp3cKO E13 . 5 cochlear sections reveals the presence of numerous aggresome - like structures in Elp3 - deficient SGNs . Scale bar = 100µm . ( B ) Transmission electron micrographs from E13 . 5 Elp3cKO SGNs confirm the presence of apoptotic neurons ( marked by a red arrowhead ) and a large electron dense juxtanuclear structures typical of aggresome ( dashed circle ) . Scale bars = 10µm and 2µm ( last panel ) . ( C ) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae ( n = 8 / 6 for WT / KO ; unpaired t - test two - tailed ; p = 0 . 0001 / 0 . 0015 / 0 . 0010 ; t = 9 . 535 / 4 . 090 / 4 . 307 ; DF = 12 / 12 / 12 for Elp3 , Chop and Chac1 respectively ; mean ± SEM presented ) indicating that the pro - apoptotic arm of the unfolded protein response ( UPR ) is activated in Elp3 - deficient SGNs . ( D - E ) Proteostat staining of whole - mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3 . Orthogonal slices ( Orthog ) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs . Scale bars = 5µm . # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 . ( F ) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3 , confirming proteostasis disruption . ( G ) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti ( n = 3 animals per genotype ; unpaired t - test two - tailed ; p = 0 . 0003 / 0 . 4296 / 0 . 056 ; t = 11 . 62 / 0 . 8778 / 2 . 662 ; DF = 4 / 4 / 4 for Elp3 , Chop and Chac1 respectively ; mean ± SEM presented ) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR - mediated apoptosis .

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

Figure 3(A) Proteostat staining of WT and Elp3cKO E13.5 cochlear sections reveals the presence of numerous aggresome-like structures in Elp3-deficient SGNs. Scale bar =100µm.(B) Transmission electron micrographs from E13.5 Elp3cKO SGNs confirm the presence of apoptotic neurons (marked by a red arrowhead) and a large electron dense juxtanuclear structures typical of aggresome (dashed circle). Scale bars =10µm and 2µm (last panel).(C) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae (n=8/6 for WT/KO; unpaired t-test two-tailed; p=0.0001/0.0015/0.0010; t=9.535/4.090/4.307; DF=12/12/12 for Elp3, Chop and Chac1 respectively; mean±SEM presented) indicating that the pro-apoptotic arm of the unfolded protein response (UPR) is activated in Elp3-deficient SGNs.(D-E) Proteostat staining of whole-mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs. Scale bars =5µm. #IHC; *OHC1; **OHC2; ***OHC3.(F) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3, confirming proteostasis disruption.(G) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti (n=3 animals per genotype; unpaired t-test two-tailed; p=0.0003/0.4296/0.056; t=11.62/0.8778/2.662; DF=4/4/4 for Elp3, Chop and Chac1 respectively; mean±SEM presented) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR-mediated apoptosis.


Figure 4 ( A ) Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity , as misaligned HCs and dysmorphic stereociliary bundles are visible ( red arrows and white arrowheads , respectively ) . Scale bars = 5µ ( B ) Surface view of P1 actin - stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio - lateral axis ( M - L ) or misshaped . Scale bars = 5µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 ( C ) Elp3cKOs exhibit a broader distribution of hair cell orientation . The angles of deviation between the stereociliary bundle and the medio - lateral axis are plotted in rose diagrams , for each OHC row . Whereas the majority of WT bundles are pointing to the most lateral region of the tissue ( within a range of 10 ° ) , many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation ( WT / Elp3cKO OHC1 : n = 99 / 99 , p > 0 . 1 , DF = 99 ; OHC2 : n = 99 / 100 , p < 0 . 001 , DF = 99 ; OHC3 : n = 99 / 98 , p < 0 . 001 , DF = 98 ; pooled data from 6 animals per genotype ; Watson U2 test ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( D ) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae ( scale bar = 2µm ) illustrating the misshaped stereociliary bundles and the shorter kinocilium ( false coloured in pink ) of Elp3cKO HCs . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( E ) Graph presents the mean angle of HC bundle ( n = 6 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , F = 13 . 69 , DF = 7 ; mean ± SD ) . Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( F ) Elp3cKO HCs possess a significantly larger bare zone ( WT / KO IHC : n = 23 / 24 cells ; OHC1 : n = 27 / 27 ; OHC2 : n = 27 / 29 ; OHC3 : n = 30 / 29 ; cumulative data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , * p < 0 . 05 F = 16 . 72 , DF = 7 ; mean ± SD ) . Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( G ) Elp3cKO HCs possess a significantly smaller medial zone ( WT / KO IHC : n = 23 / 24 cells ; OHC1 : n = 27 / 27 ; OHC2 : n = 27 / 29 ; OHC3 : n = 27 / 29 ; cumulative data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , * p < 0 . 05 , F = 63 . 3 , DF = 7 ; mean ± SD ) . ( H ) Kinocilia are shorter in Elp3cKO HCs ( WT / KO IHC : n = 15 / 15 ; OHC1 : n = 30 / 32 ; OHC3 : n = 31 / 30 ; OHC3 : n = 27 / 30 ; data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test , * * * p < 0 . 001 , * * p < 0 . 01F = 19 . 6 , DF = 7 ; mean ± SD ) . ( I ) High magnification of representative HCs from P1 WT and Elp3cKO cochlea stained for F - actin and gamma - tubulin ( white ) . The basal body of kinocilia is shifted centrally at the apical surface of Elp3cKO HCs . Scale bars = 2µm . Polar plotting of kinocilium basal body position for each row of HCs of WT and Elp3cKO ( WT / KO IHC : n = 42 / 32 ; OHC1 : n = 47 / 48 ; OHC2 : n = 47 / 45 ; OHC3 : n = 44 / 44 ; cumulative data collected from 3 animals per genotype ) . Cellular orientations were tested using Watson U2 test ( p < 0 . 001 between WT / Elp3cKO , for all HC rows ) and the mean vector lengths , determined from the cell centre to the basal body , were compared using t - tests ( p < 0 . 001 between WT / Elp3cKO , for all HC rows ) . The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with LGN ( J ) and F - actin . The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with Gαi3 ( K ) and F - actin . The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with aPKC ( L ) and F - actin . The enrichment of these apical polarity regulators at the medial ( L ) edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

Figure 4(A) Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity, as misaligned HCs and dysmorphic stereociliary bundles are visible (red arrows and white arrowheads, respectively). Scale bars=5µ(B) Surface view of P1 actin-stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio-lateral axis (M-L) or misshaped. Scale bars=5µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3(C) Elp3cKOs exhibit a broader distribution of hair cell orientation. The angles of deviation between the stereociliary bundle and the medio-lateral axis are plotted in rose diagrams, for each OHC row. Whereas the majority of WT bundles are pointing to the most lateral region of the tissue (within a range of 10°), many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation (WT/Elp3cKO OHC1: n=99/99, p>0.1, DF=99; OHC2: n=99/100, p<0.001, DF=99; OHC3: n=99/98, p<0.001, DF=98; pooled data from 6 animals per genotype; Watson U2 test). Data information: #IHC; *OHC1; **OHC2; ***OHC3Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (D) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae (scale bar=2µm) illustrating the misshaped stereociliary bundles and the shorter kinocilium (false coloured in pink) of Elp3cKO HCs. Data information: #IHC; *OHC1; **OHC2; ***OHC3Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (E) Graph presents the mean angle of HC bundle (n=6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, F=13.69, DF=7; mean±SD).Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (F) Elp3cKO HCs possess a significantly larger bare zone (WT/KO IHC: n= 23/24 cells; OHC1: n=27/27; OHC2: n=27/29; OHC3: n=30/29; cumulative data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, *p<0.05 F=16.72, DF=7; mean±SD).Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (G) Elp3cKO HCs possess a significantly smaller medial zone (WT/KO IHC: n= 23/24 cells; OHC1: n=27/27; OHC2: n=27/29; OHC3: n=27/29; cumulative data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, *p<0.05, F=63.3, DF=7; mean±SD).(H) Kinocilia are shorter in Elp3cKO HCs (WT/KO IHC: n=15/15; OHC1: n=30/32; OHC3: n=31/30; OHC3: n=27/30; data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, **p<0.01F=19.6, DF=7; mean±SD).(I) High magnification of representative HCs from P1 WT and Elp3cKO cochlea stained for F-actin and gamma-tubulin (white). The basal body of kinocilia is shifted centrally at the apical surface of Elp3cKO HCs. Scale bars=2µm. Polar plotting of kinocilium basal body position for each row of HCs of WT and Elp3cKO (WT/KO IHC: n= 42/32; OHC1: n= 47/48; OHC2: n= 47/45; OHC3: n= 44/44; cumulative data collected from 3 animals per genotype). Cellular orientations were tested using Watson U2 test (p<0.001 between WT/Elp3cKO, for all HC rows) and the mean vector lengths, determined from the cell centre to the basal body, were compared using t-tests (p<0.001 between WT/Elp3cKO, for all HC rows).The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with LGN (J) and F-actin. The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with Gαi3 (K) and F-actin. The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with aPKC (L) and F-actin. The enrichment of these apical polarity regulators at the medial (L) edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


Figure 5 ( A - B ) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown . ( A ) HEK293T cells transfected with siRNAs for Elp3 , Alkbh8 or control and immunolabelled with LGN , acetylated alpha - tubulin and DAPI . Scale bar = 5µm . ( B ) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified ( siRNA Control / Elp3 / Alkbh8 : n = 27 / 30 / 27 ; pooled from 3 independent experiments ; mean ± SEM ) . ( C - D ) The length of the primary cilium is reduced in non - dividing HEK293T cells upon Elp3 or Albh8 knockdown . ( C ) Control , Elp3 or Alkbh8 siRNA - treated HEK293T cells were immunolabelled for basal body marker ALMS1 , axoneme marker Arl13b and DAPI . Scale bar = 5µm . ( D ) Primary cilium length of non - dividing HEK293T was measured and pooled data were plotted ( siRNA Control / Elp3 / Alkbh8 : n = 94 / 81 / 90 ; data collected from 3 independent experiments ; One - way ANOVA , Tukey ' s multiple comparisons test , * * * p < 0 . 001 , F = 21 . 46 , DF = 4 ; minimum , first quartile , median , third quartile and maximum presented ) . ( E - F ) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells ( n = 3 ; One - way ANOVA , Tukey ' s multiple comparisons test , * * p < 0 . 01 , * p < 0 . 05 , F = 16 . 51 , DF = 2 ; mean ± SD ) . ( G ) Increased formation of aggresome - like structures in Elp3 - or Alkbh8 - depleted HEK293T . Aggresomes ( red ) ; DAPI ( blue ) . Enlarged images depict examples of non - dividing ( left ) and dividing ( right ) cells . Scale bar = 10µm . Inducing aggresomes with MG - 132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells . ( H ) HEK293T cells treated with DMSO vehicle or MG - 132 and stained for aggresomes ( left panels ) or LGN , F - actin and DAPI ( green , red and blue , respectively in right panels ) . Scale bar = 10µm . Inducing aggresomes with MG - 132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells . ( I ) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells . Mean fluorescence values per percentage of cell length with SEM are plotted . Fluorescence intensity profiles collected from 3 independent experiments ( DMSO / MG - 132 : n = 24 / 24 ) . ( J ) Proteostat staining of MG132 - treated whole - mounted P0 organs of Corti reveals the presence of aggresome - like structures in HCs . Orthogonal slices ( Orthog ) reveal aggregated proteins localize at the apical surface of MG132 - treated HCs . Scale bars = 5µm . # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 ( K ) Localization of LGN at the lateral apical surface of E16 . 5 WT HCs is disrupted by MG - 132 . LGN ( white / green ) and F - actin ( red ) . Scale bars = 5µm ; magnified views = 2µm .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-B) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown. (A) HEK293T cells transfected with siRNAs for Elp3, Alkbh8 or control and immunolabelled with LGN, acetylated alpha-tubulin and DAPI. Scale bar=5µm. (B) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified (siRNA Control/Elp3/Alkbh8: n=27/30/27; pooled from 3 independent experiments; mean±SEM).(C-D) The length of the primary cilium is reduced in non-dividing HEK293T cells upon Elp3 or Albh8 knockdown. (C) Control, Elp3 or Alkbh8 siRNA-treated HEK293T cells were immunolabelled for basal body marker ALMS1, axoneme marker Arl13b and DAPI. Scale bar=5µm. (D) Primary cilium length of non-dividing HEK293T was measured and pooled data were plotted (siRNA Control/Elp3/Alkbh8: n=94/81/90; data collected from 3 independent experiments; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, F=21.46, DF=4; minimum, first quartile, median, third quartile and maximum presented).(E-F) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells (n=3; One-way ANOVA, Tukey's multiple comparisons test, **p<0.01, *p<0.05, F=16.51, DF=2; mean±SD).(G) Increased formation of aggresome-like structures in Elp3- or Alkbh8-depleted HEK293T. Aggresomes (red); DAPI (blue). Enlarged images depict examples of non-dividing (left) and dividing (right) cells. Scale bar=10µm.Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (H) HEK293T cells treated with DMSO vehicle or MG-132 and stained for aggresomes (left panels) or LGN, F-actin and DAPI (green, red and blue, respectively in right panels). Scale bar=10µm.Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (I) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells. Mean fluorescence values per percentage of cell length with SEM are plotted. Fluorescence intensity profiles collected from 3 independent experiments (DMSO/MG-132: n= 24/24).(J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3(K) Localization of LGN at the lateral apical surface of E16.5 WT HCs is disrupted by MG-132. LGN (white/green) and F-actin (red). Scale bars=5µm; magnified views=2µm.


Figure 6LGN and Gαi3 protein levels are not affected by Elp3 loss . ( A ) Western blots with total lysates from control and Elp3 siRNA - treated HEK293T cells and corresponding quantifications of normalized LGN , Gαi3 and Elp3 levels ( n = 6 from 2 independent cultures ; Unpaired two - tailed t - test , LGN : p = 0 . 749 , t = 0 . 328 , DF = 10 , Gαi3 : p = 0 . 275 , t = 1 . 155 , DF = 10 , Elp3 : * * * p = 0 . 0003 , t = 5 . 438 , DF = 10 , mean ± SEM ) . LGN and Gαi3 protein levels are not affected by Elp3 loss . ( B ) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels ( n = 3 , each lysate obtained from cochleae pooled from 2 animals ; Unpaired two - tailed t - test , LGN : p = 0 . 586 , t = 0 . 591 , DF = 4 , Gαi3 : p = 0 . 963 , t = 0 . 0491 , DF = 4 , mean ± SEM ) . ( C ) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion . HEK293T transfected with Elp3 siRNA were co - stained for aggresomes , Dapi and LGN or Gαi3 . Scale bar = 5µm . Microtubular transport speed is reduced by the presence of aggresome - like structures in HEK293T cells and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HEK293T cells labelled with LysoTracker ® . ( D ) : DMSO / MG - 132 / MG - 132 + 4PBA n = 555 / 337 / 453 , pooled from 3 experiments , * * * p < 0 . 001 , * * p < 0 . 01 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum . Microtubular transport speed is reduced by the presence of aggresome - like structures in HEK293T cells and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HEK293T cells labelled with LysoTracker ® . ( E ) : siCtl / + 4PBA n = 622 / 677 ; siElp3 / + 4PBA n = 674 / 577 ; siAlkbh8 / + 4PBA n = 460 / 484 , pooled from 4 experiments , * * * p < 0 . 001 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum . Microtubular transport speed is reduced by the presence of aggresome - like structures in cultured HCs , and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HCs , at the level of their apical surface labelled with LysoTracker ® . ( F ) : DMSO / MG - 132 / MG - 132 + 4PBA n = 210 / 225 / 266 , pooled from 3 experiments , * * * p < 0 . 001 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum . Microtubular transport speed is reduced by the presence of aggresome - like structures in cultured HCs , and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HCs , at the level of their apical surface labelled with LysoTracker ® . ( G ) : WT / WT + 4PBA / Elp3cKO / Elp3cKO + 4PBA n = 732 / 763 / 730 / 738 , pooled from 4 OCs per condition , * * * p < 0 . 001 , * * p < 0 . 01 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Figure 6LGN and Gαi3 protein levels are not affected by Elp3 loss. (A) Western blots with total lysates from control and Elp3 siRNA-treated HEK293T cells and corresponding quantifications of normalized LGN, Gαi3 and Elp3 levels (n=6 from 2 independent cultures; Unpaired two-tailed t-test, LGN: p=0.749, t=0.328, DF=10, Gαi3: p=0.275, t=1.155, DF=10, Elp3: ***p=0.0003, t=5.438, DF=10, mean±SEM).LGN and Gαi3 protein levels are not affected by Elp3 loss. (B) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels (n=3, each lysate obtained from cochleae pooled from 2 animals; Unpaired two-tailed t-test, LGN: p=0.586, t=0.591, DF=4, Gαi3: p=0.963, t=0.0491, DF=4, mean±SEM).(C) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion. HEK293T transfected with Elp3 siRNA were co-stained for aggresomes, Dapi and LGN or Gαi3. Scale bar=5µm.Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (D): DMSO/MG-132/MG-132+4PBA n=555/337/453, pooled from 3 experiments, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (E): siCtl/+4PBA n=622/677; siElp3/+4PBA n=674/577; siAlkbh8/+4PBA n=460/484, pooled from 4 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (F): DMSO/MG-132/MG-132+4PBA n=210/225/266, pooled from 3 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (G): WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA n=732/763/730/738, pooled from 4 OCs per condition, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Figure 7Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( A ) LGN and F - actin immunostainings of E16 . 5 + 1DIV OCs reveal that MG - 132 perturbs LGN distribution at the apical surface of HCs , while co - treatment with 4 - PBA ameliorates its enrichment at the lateral edge . Scale bars = 5µm ; magnified views = 2µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( B ) Acetylated alpha - tubulin immunostaining reveals that the kinocilium is centrally shifted by MG - 132 and partially restored by 4 - PBA in E16 . 5 + 1DIV HCs ( yellow arrowheads = kinocilium base ; scale bar = 2µm ) . Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( C ) Kinocilium position plotted as a fraction of HC diameter for each HC row ( DMSO / MG - 132 / MG - 132 + 4PBA IHC : n = 57 / 67 / 53 , * * * p < 0 . 001 , F = 34 . 52 , DF = 2 ; OHC1 : n = 61 / 66 / 53 , * * * p < 0 . 001 , F = 57 . 88 , DF = 2 ; OHC2 : n = 55 / 73 / 58 , * * * p < 0 . 001 , F = 124 . 7 , DF = 2 ; OHC3 : n = 48 / 66 / 43 , * * * p < 0 . 001 , F = 67 . 16 , DF = 2 ; pooled from 4 explants per condition ; mean ± SD ; One - way ANOVA with Tukey ' s multiple comparisons test ) . In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( D ) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration . Scale bars = 5µm ; magnified views = 2µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( E ) Acetylated alpha - tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4 - PBA ( yellow arrowheads = base of kinocilium ; scale bar = 2µm ) . In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( F ) Kinocilium position plotted as a fraction of HC diameter for each HC row ( WT / WT + 4PBA / Elp3cKO / Elp3cKO + 4PBA IHC : n = 35 / 31 / 35 / 36 , p = 0 . 6812 , F = 4 . 510 , DF = 3 ; OHC1 : n = 36 / 32 / 37 / 40 , * * * p < 0 . 001 , * * p < 0 . 01 , F = 27 . 60 , DF = 3 ; OHC2 : n = 35 / 33 / 38 / 42 , * * * p < 0 . 001 , F = 76 . 62 , DF = 3 ; OHC3 : n = 36 / 32 / 38 / 39 , * * * p < 0 . 001 , * p < 0 . 05 , F = 27 . 86 , DF = 3 ; pooled from 3 animals per condition ; mean ± SD ; One - way ANOVA with Tukey ' s multiple comparisons test ) .

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

Figure 7Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm).Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm).In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).