Figure 4A . The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition . HEK293T cells were transfected with Flag - Merlin and LRP6 - EGFP . After transfection , cells were incubated in serum - free medium for overnight with Bafilomycin A1 ( 20 nM ) . The cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting with the indicated antibodies . WCL , whole - cell lysates . The interaction between endogenous Merlin and LRP6 was reduced under serum starvation . HEK293T cells were incubated in serum - free medium for 4 h with Bafilomycin A1 ( 100 nM ) . The cell lysates were immunoprecipitated with anti - LRP6 antibody and analyzed by immunoblotting with the indicated antibodies . WCL , whole - cell lysates . The interaction between endogenous Merlin and LRP6 was reduced under serum starvation . HEK293T cells were incubated in serum - free medium for 4 h with Bafilomycin A1 ( 100 nM ) . The cell lysates were immunoprecipitated with anti - Merlin antibody and analyzed by immunoblotting with the indicated antibodies . WCL , whole - cell lysates . D . The interaction between overexpressed Merlin and LATS1 was increased under serum starvation . HEK293T cells were transfected with Myc - LATS1 and EGFP - Merlin . After transfection , cells were incubated in serum - free medium for overnight and then were lysed . The cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates . E . Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1 . HEK293T cells were transfected with Myc - LATS1 , EGFP - Merlin and LRP6 siRNA . Cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates . F . Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1 . HEK293T cells were transfected with siRNA for GFP or LRP6 . After transfection , cells were lysed and the cell lysates were immunoprecipitated with anti - Merlin antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.D. The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc-LATS1 and EGFP-Merlin. After transfection, cells were incubated in serum-free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.E. Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc-LATS1, EGFP-Merlin and LRP6 siRNA. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.F. Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


Figure 5A . Endogenous LRP6 and OGT interact with each other . HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti - LRP6 antibody and blotted with OGT . WCL , whole - cell lysates . B . LRP6 is O - GlcNAcylated . LRP6 - EGFP - transfected HEK293T cells were treated in Thiamet G ( 30 μM ) or OSMI - 1 ( 50 μM ) for 6 h with Bafiolmycin A1 ( 100nM ) . After treatment , cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates . C . LRP6 is O - GlcNAcylated . Cell lysates from HEK293T cells were incubated with sWGA - beads in the absence or presence of free GlcNAc ( 25 mM ) for 3 h . After incubation , sWGA - beads were precipitated and analyzed by immunoblotting . WCL , whole - cell lysates . D . Blocking of O - GlcNAcylation reduced the amount of LRP6 . HEK293 cells were treated with OSMI - 1 ( 50 μM ) and Bafilomycin A1 ( 100 nM ) for 6 h . Cells were lysed and the cell lysates were analyzed by immunoblotting . E . The O - GlcNAcylation on LRP6 was reduced under serum starvation condition . HEK293T cells were transfected with LRP6 - EGFP . Cells were incubated in serum - free medium with Bafilomycin A1 ( 100 nM ) for 4 h . After incubation , cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates . The ratio of O - GlcNAc / EGFP from three independent immunoblots was quantified ( biological replicates , bottom panel ) . * P < 0 . 05 . Error bars indicate standard deviation of biological triplicate measurements . Student ' s t - test was used for statistical analysis . F . Increase of O - GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin . LRP6 - EGFP and Flag - Merlin transfected HEK293T cells were incubated in serum - free medium with Bafilomycin A1 ( 20 nM ) and Thiamet G ( 30μM ) overnight . Cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates . G . Enhancement of global O - GlcNAcylation blocked the serum starvation - mediated increase of interaction between overexpressed LATS1 and Merlin . HEK293T cells were transfected with Myc - LATS1 and Flag - Merlin . After transfection , cells were incubated in serum - free medium with Thiamet G ( 30μM ) and Bafilomycin A1 ( 20nM ) overnight . Cells were lysed and the cell lysates were immunoprecipitated with anti - Myc antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

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

Figure 5A. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti-LRP6 antibody and blotted with OGT. WCL, whole-cell lysates.B. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


Figure 6A . Nutrient starvation reduced the level of LRP6 in mouse liver . Mice were starved for 36 h and liver tissues were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / Vinculin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * * P < 0 . 01 . Student ' s t - test was used for statistical analysis . B . Refeeding of nutrients rescued LRP6 in mouse liver . Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / Vinculin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 . Student ' s t - test was used for statistical analysis . C . The level of LRP6 was not reduced during nutrient starvation in mouse brain . Mice were starved and brain tissues were analyzed by immunoblotting . D . LRP6 was O - GlcNAcylated in mouse liver . Liver tissues were incubated with sWGA - beads in the absence or presence of free GlcNAc ( 25 mM ) for 3 h . After incubation , sWGA - beads were precipitated and analyzed by immunoblotting . WCL , whole - cell lysates . E . O - GlcNAcylation of LRP6 was reduced during starvation in mouse liver . LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel . Liver tissues were incubated in sWGA - beads for 3 h . After incubation , sWGA - beads were precipitated and analyzed by immunoblotting . WCL , whole - cell lysates .

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

Figure 6A. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t-test was used for statistical analysis.B. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting.D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.E. O-GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA-beads for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


Figure 1Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc - / - mice ( A ) . Quantification of Gadd45a protein expression , β - actin protein level was used as the loading control ( n = 7 mice per genotype ) , two independent experiments were carried out ( B ) . Quantification of Gadd45a - ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc - / - MEF cells ( n = 3 for each genotype ) . Dot - bot assay following Gadd45a - ChIP in WT and G3Terc - / - MEF cells . Kaplan - Meier survival curves of the mice with indicated genotypes ( WT , n = 44 ; Gadd45a - / - , n = 48 ; G3Terc - / - , n = 37 ; G3 - dKO , n = 41 ) . P value comparing the survival of G3Terc - / - with G3 - dKO mice is calculated with the log - rank test . The average body weight of mice with indicated genotypes at indicated time points ( G3 - dKO Female ( n = 19 ) vs G3Terc - / - Female ( n = 17 ) , P = 0 . 004 ; G3 - dKO Male ( n = 14 ) vs G3Terc - / - Male ( n = 14 ) , P = 0 . 0038 ) . P values are calculated with log - rank test . Whole mount images of colon samples from 7 - month old mice with indicated genotypes ( G ) . Quantification on density of colon crypts ( H ) ( n = 5 mice per group ) . H & E - stained small intestine sections from 7 - month old mice with indicated genotypes ( I ) . Quantification of the number of small intestine basal crypts ( J ) ( n = 5 - 6 mice per group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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 1Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc-/- mice (A). Quantification of Gadd45a protein expression, β-actin protein level was used as the loading control (n=7 mice per genotype), two independent experiments were carried out (B).Quantification of Gadd45a-ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc-/- MEF cells (n=3 for each genotype).Dot-bot assay following Gadd45a-ChIP in WT and G3Terc-/- MEF cells.Kaplan-Meier survival curves of the mice with indicated genotypes (WT, n=44; Gadd45a-/-, n=48; G3Terc-/-, n=37; G3-dKO, n=41). P value comparing the survival of G3Terc-/- with G3-dKO mice is calculated with the log-rank test.The average body weight of mice with indicated genotypes at indicated time points (G3-dKO Female (n=19) vs G3Terc-/- Female (n=17), P=0.004; G3-dKO Male (n=14) vs G3Terc-/- Male (n=14), P=0.0038). P values are calculated with log-rank test.Whole mount images of colon samples from 7-month old mice with indicated genotypes (G). Quantification on density of colon crypts (H) (n=5 mice per group).H&E-stained small intestine sections from 7-month old mice with indicated genotypes (I). Quantification of the number of small intestine basal crypts (J) (n=5-6 mice per group).


Figure 2Olfm4 - positive intestinal stem cells ( indicated by arrows ) in small intestine sections ( A ) . Quantification of intestinal stem cell ( Olfm4 + ) number per 100 μm ( n = 4 - 5 mice per group ) of small intestine sections ( B ) . Representative images of small intestine crypt culture ( day 2 ) . Arrows mark the surviving cysts ( C ) . Quantification of numbers of cysts per culture well ( D ) ( n = 4 - 5 mice / genotype ) . In vitro crypt culture ( day 8 ) from small intestine crypts isolated from mice with indicated genotypes . Arrows mark the crypt - like structure ( E ) . The number of organoids per culture wells ( n = 4 - 5 mice per group ) . Two hundred crypts are seeded per well ( F ) . Representative images of TUNEL staining in small intestine sections . Arrows indicate TUNEL positive cells ( G ) . Quantification of TUNEL positive cells per crypt ( n = 5 mice per group ) ( H ) . Representative images of phosphorylated histone 3 ( pH3 ) antibody staining in mouse small intestine . Arrows indicate pH3 positive cells ( I ) . Quantification of pH3 positive crypt cells per 100 μm of small intestines ( n = 4 - 6 mice per group ) ( J ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Olfm4-positive intestinal stem cells (indicated by arrows) in small intestine sections (A). Quantification of intestinal stem cell (Olfm4+) number per 100 μm (n=4-5 mice per group) of small intestine sections (B).Representative images of small intestine crypt culture (day 2). Arrows mark the surviving cysts (C). Quantification of numbers of cysts per culture well (D)(n=4-5 mice/genotype).In vitro crypt culture (day 8) from small intestine crypts isolated from mice with indicated genotypes. Arrows mark the crypt-like structure (E). The number of organoids per culture wells (n=4-5 mice per group). Two hundred crypts are seeded per well (F).Representative images of TUNEL staining in small intestine sections. Arrows indicate TUNEL positive cells (G). Quantification of TUNEL positive cells per crypt (n=5 mice per group) (H).Representative images of phosphorylated histone 3 (pH3) antibody staining in mouse small intestine. Arrows indicate pH3 positive cells (I). Quantification of pH3 positive crypt cells per 100 μm of small intestines (n=4-6 mice per group) (J).


Figure 3Representative images of immunostaining for p53 on small intestine sections . Arrows indicate p53 - positive cells ( A ) . Quantification of the percentage of p53 positive cells per crypt ( n = 5 - 6 mice per group ) ( B ) . Representative images of γH2AX antibody staining on small intestine paraffin sections from WT , Gadd45a - / - , G3Terc - / - , and G3 - dKO mice ( 7 months old ) . Arrows indicate γH2AX positive nuclei ( C ) . Quantification of the percentage of γH2AX positive cells per small intestine crypt ( n = 4 - 6 mice per group ) ( D ) . Representative images of small intestine crypt sections stained with antibody against pATM ( Ser - 1981 ) ( E ) . Quantification of frequencies of pATM ( Ser - 1981 ) positive crypt cells ( n = 4 - 6 of 7 - month old mice per genotype ) ( F ) . Representative image of the anaphase bridge ( indicated with arrows ) in the basal crypt of G3Terc - / - mice ( G ) . The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population ( n = 4 - 6 mice per genotype ) ( H ) . Heat map indicating the relative expression levels of DDR and cell cycle - related gene transcripts in MEFs from indicated genotypes ( Data are extracted from RNAseq datasets ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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 3Representative images of immunostaining for p53 on small intestine sections. Arrows indicate p53-positive cells (A). Quantification of the percentage of p53 positive cells per crypt (n=5-6 mice per group) (B).Representative images of γH2AX antibody staining on small intestine paraffin sections from WT, Gadd45a-/-, G3Terc-/-, and G3-dKO mice (7 months old). Arrows indicate γH2AX positive nuclei (C). Quantification of the percentage of γH2AX positive cells per small intestine crypt (n=4-6 mice per group) (D).Representative images of small intestine crypt sections stained with antibody against pATM (Ser-1981)(E). Quantification of frequencies of pATM (Ser-1981) positive crypt cells (n=4-6 of 7-month old mice per genotype) (F).Representative image of the anaphase bridge (indicated with arrows) in the basal crypt of G3Terc-/- mice (G). The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population (n=4-6 mice per genotype) (H).Heat map indicating the relative expression levels of DDR and cell cycle-related gene transcripts in MEFs from indicated genotypes (Data are extracted from RNAseq datasets).


Figure 4CpG island methylation status of subtelomeric regions on chromosome 1q . gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing . Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands , respectively , at each position ( A ) . Quantification of subtelomeric CpG island methylation ( B ) . Telomeric quantification and dot - bot assay of tri - methyl H3K9 ( H3K9me3 ) - ChIP in indicated genotypesTelomeric quantification and dot - bot assay of acetylated H3K9 ( H3K9ac ) - ChIP in indicated genotypesTelomeric quantification and dot - bot assay of HP1a - ChIP in indicated

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

Figure 4CpG island methylation status of subtelomeric regions on chromosome 1q. gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing. Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands, respectively, at each position (A). Quantification of subtelomeric CpG island methylation (B).Telomeric quantification and dot-bot assay of tri-methyl H3K9 (H3K9me3)-ChIP in indicated genotypesTelomeric quantification and dot-bot assay of acetylated H3K9 (H3K9ac)-ChIP in indicated genotypesTelomeric quantification and dot-bot assay of HP1a-ChIP in indicated genotypes


Figure 5Representative images of small intestine organoids from WT and G3Terc - / - mice incubated with or without APE1 inhibitor ( CRT0044876 , 10μM ) treatment . Arrows indicate the crypt - like structure in organoid cultures ( A ) . Quantification of organoid number ( per well ) and size ( in 104 μm2 ) ( n = 3 - 4 mice per genotype / treatment ) ( B - C ) . Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc - / - organoid cultures with or without APE1 inhibitor ( CRT0044876 , 10μM ) ( DNAs are stained with DAPI ) ( D ) . Quantification of γH2AX positive cells ( n = 3 - 4 mice per group ) ( E ) . Telomeric DNA quantification and dot - blot assay of H3K9me3 - ChIPTelomeric DNA quantification and dot - blot assay of H3K9ac - ChIPTelomeric DNA quantification and dot - blot assay of HP1a - ChIPRepresentative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor ( CRT0044876 , CRT ) . Arrows indicate Olfm4 positive intestine stem cells ( I ) . Quantification of Olfm4 positive intestine stem cells ( n = 4 mice per group ) ( J ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Representative images of small intestine organoids from WT and G3Terc-/- mice incubated with or without APE1 inhibitor (CRT0044876, 10μM) treatment. Arrows indicate the crypt-like structure in organoid cultures (A). Quantification of organoid number (per well) and size (in 104 μm2) (n=3-4 mice per genotype/treatment) (B-C).Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc-/- organoid cultures with or without APE1 inhibitor (CRT0044876, 10μM) (DNAs are stained with DAPI) (D). Quantification of γH2AX positive cells (n=3-4 mice per group) (E).Telomeric DNA quantification and dot-blot assay of H3K9me3-ChIPTelomeric DNA quantification and dot-blot assay of H3K9ac-ChIPTelomeric DNA quantification and dot-blot assay of HP1a-ChIPRepresentative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor (CRT0044876, CRT). Arrows indicate Olfm4 positive intestine stem cells (I). Quantification of Olfm4 positive intestine stem cells (n=4 mice per group) (J).


Figure 6The knockdown efficiency of shRNA - GADD45A in human WI38 fibroblasts . The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A . Representative images of β - gal staining in human fibroblast cultures with control and shRNA - GADD45A treatments at passage 54 ( C ) . Quantification of the percentage of β - gal positive cells in human fibroblasts with control and shRNA - GADD45A treatments ( duplicates per group ) ( D ) . Representative images of GADD45A and γH2AX staining in colon sections from young ( 35 - 50 y / o ) and old ( 75 - 85 y / o ) people ( E ) . Quantification of the percentage of GADD45A positive cells in young and old human colon tissues ( n = 8 per age group ) ( F ) . Quantification of the percentage of γH2AX positive cells in young and old human colon tissues ( n = 6 per age group ) ( G ) .

O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6The knockdown efficiency of shRNA-GADD45A in human WI38 fibroblasts.The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A.Representative images of β-gal staining in human fibroblast cultures with control and shRNA-GADD45A treatments at passage 54 (C). Quantification of the percentage of β-gal positive cells in human fibroblasts with control and shRNA-GADD45A treatments (duplicates per group) (D).Representative images of GADD45A and γH2AX staining in colon sections from young (35-50 y/o) and old (75-85 y/o) people (E). Quantification of the percentage of GADD45A positive cells in young and old human colon tissues (n=8 per age group) (F). Quantification of the percentage of γH2AX positive cells in young and old human colon tissues (n=6 per age group) (G).


Figure 1Body weights of WT and Rcan1 - KO mice fed normal chow diet ( NC ) for 20 weeks starting at 8 - 10 weeks of age ( male , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . Body weights of WT and Rcan1 - KO mice fed a high - fat diet ( HFD ) for 20 weeks starting at 8 - 10 weeks of age ( male , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . C Food consumption per gram of body weight gained ( g / g ∆ BW ) over the first 15 weeks on NC or HFD , starting at 8 weeks of age ( male , n = 9 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . Cholesterol remaining in the feces of WT and Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( males , n = 4 over 3 days ) . Cholesterol content of NC and the HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . triglyceride remaining in the feces of WT and Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( males , n = 4 over 3 days ) . triglyceride content of NC and the HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . MRI measures of total body weight , percentage lean mass , and percent fat mass prior to and after 6 weeks on the HFD ( males , n = 5 ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . MRI image of WT and KO males following 8 weeks on a HFD . Yellow delineates visceral WAT . Pink delineates subcutaneous WAT . Quantification graphed on the right . Data information : values shown are mean ± SEM * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . Representative H & E staining of gWAT from WT and KO males after 25 weeks on NC or HFD . Scale bar = 100 μm . K Quantification of cross - sectional area ( CSA ) of adipocytes in K ( n = 3 animals , 3 images / each , 75 - 100 adipocytes ) . Scale bar = 100 μm . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) . L Transcript levels for inflammatory markers Monocyte chemoattractant protein - 1 ( Mcp - 1 ) , macrophage antigen F4 / 80 ( F480 ) , tumor necrosis factor - alpha ( Tnfα ) , and tumor necrosis factor - beta ( Tnfβ ) in gWAT of WT and KO after 25 weeks on NC or HFD ( males , n = 4 - 5 ) . Transcript levels were normalized to 18S Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Body weights of WT and Rcan1-KO mice fed normal chow diet (NC) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).Body weights of WT and Rcan1-KO mice fed a high-fat diet (HFD) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).C Food consumption per gram of body weight gained (g/g ∆BW) over the first 15 weeks on NC or HFD, starting at 8 weeks of age (male, n=9 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).Cholesterol remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). Cholesterol content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).triglyceride remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). triglyceride content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).MRI measures of total body weight, percentage lean mass, and percent fat mass prior to and after 6 weeks on the HFD (males, n=5). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).MRI image of WT and KO males following 8 weeks on a HFD. Yellow delineates visceral WAT. Pink delineates subcutaneous WAT. Quantification graphed on the right. Data information: values shown are mean ±SEM * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).Representative H&E staining of gWAT from WT and KO males after 25 weeks on NC or HFD. Scale bar = 100 μm. K Quantification of cross-sectional area (CSA) of adipocytes in K (n=3 animals, 3 images/each, 75-100 adipocytes). Scale bar = 100 μm. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).L Transcript levels for inflammatory markers Monocyte chemoattractant protein-1 (Mcp-1), macrophage antigen F4/80 (F480), tumor necrosis factor-alpha (Tnfα), and tumor necrosis factor-beta (Tnfβ) in gWAT of WT and KO after 25 weeks on NC or HFD (males, n=4-5). Transcript levels were normalized to 18S Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Figure 2A , B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD , with quantification of relative stain intensity ( n = 3 , 3 images each ) . Scale bar = 100 μm . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) . C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD . Stearoyl - Coenzyme A desaturase ( Scd1 ) , fatty acid transporter ( CD36 ) , glycerol - 3 - phosphate acyltransferase ( Gpat ) , carnitine palmitoyltransferase I ( Cpt1 ) , peroxisome proliferator - activated gamma ( PPARγ ) , pyruvate dehydrogenase acetyl - transferase ( Pdk4 ) , PPARγ coactivator 1 alpha ( PGC1α ) , diacylglycerol O - acyltransferase ( Dgat ) , and facilitated glucose transporter ( Glut4 ) . ( males , n = 4 - 5 ) . Transcript levels were normalized to 18S . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) . D - F Serum levels of free fatty acids ( FAA ) , insulin , and leptin in WT and KO after 25 weeks on NC or HFD ( males , n = 8 - 10 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) . Glucose tolerance test ( GTT ) after 25 weeks on NC or HFD . GTT was performed following an overnight fast Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) . Insulin tolerance test ( ITT ) after 25 weeks on NC or HFD . , ITT following a three hour fast ( males , n = 6 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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

Figure 2A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD. Stearoyl-Coenzyme A desaturase (Scd1), fatty acid transporter (CD36), glycerol-3-phosphate acyltransferase (Gpat), carnitine palmitoyltransferase I (Cpt1), peroxisome proliferator-activated gamma (PPARγ), pyruvate dehydrogenase acetyl-transferase (Pdk4), PPARγ coactivator 1 alpha (PGC1α), diacylglycerol O-acyltransferase (Dgat), and facilitated glucose transporter (Glut4). (males, n=4-5). Transcript levels were normalized to 18S. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).D-F Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).Glucose tolerance test (GTT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).Insulin tolerance test (ITT) after 25 weeks on NC or HFD. , ITT following a three hour fast (males, n=6). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Figure 3Oxygen consumption ( VO2 ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) . carbon dioxide production ( VCO2 ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) . respiratory exchange ratio VCO2 / VO2 ( RER ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) . physical activity of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Oxygen consumption (VO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). carbon dioxide production (VCO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). respiratory exchange ratio VCO2/VO2 (RER) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).physical activity of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


Figure 4Transcript levels for Rcan1 . 1 and Rcan1 . 4 during differentiation of 3T3 - L1 adipocytes , measured at 0 , 1 , 2 , 3 , 4 , 6 and 24 hours following the addition MDI as well as at 3 , 7 and 10 days as indicated ( n = 3 ) . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - test , unpaired , vs 0 time point . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SDB Western blot for RCAN1 . 1 , RCAN1 . 4 and tubulin ( TUB ) in whole cell extracts of 3T3 - L1 adipocytes transfected with a control si - RNA or ones targeting either Rcan1 . 4 or Rcan1 . 1 ( 20 μg per lane ) . C Representative images of 3T3 - L1 adipocytes transfected with the indicated si - RNAs prior to differentiation and stained with Oil Red O on day 10 . Scale bar = 100 μm . D Western blot for adiponectin ( ADPOQ ) released into the media during differentiation by 3T3 - L1 adipocytes transfected with the indicated si - RNAs . Ponceau stained image provides loading control . E Transcript levels for Pgc1 - α , in 3T3 - L1 adipocytes transfected with the indicated si - RNAs and assayed over 10 - days of differentiation ( n = 3 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SDRepresentative bright - field images showing lipid accumulation at 10 - days differentiation of Ing - svf cells treated with the indicated siRNA . Scale bar = 50 μm . G Transcript levels for Adrenoceptor beta 3 ( Adrb3 ) , uncoupling protein 1 ( Ucp1 ) , PPARγ coactivator 1 alpha ( PGC1α ) , and deiodinase type II ( Dio2 ) Ing - svf cultures transfected with control siRNA ( C ) or one targeting Rcan1 ( KD ) then maintained in growth media ( G ) , or differentiated for ten - days ( D ) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting ( D + ISO ) ( n = 2 in triplicate ) . * = p < 0 . 05 ; * * = p < 0 . 01 , two - way ANOVA with multiple comparisons . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SDH , I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet - derived growth factor receptor alpha - positive ( PDGFRα + ) and lineage - negative ( Lin - ) stromal vascular cells isolated by FACS sorting from sWAT of WT ( black bars ) and KO ( red bars ) . In H , isolated cells were maintained in growth media . In I , cultures were treated with MDI then differentiated for 10 days . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - test , unpaired , WT vs . KO . Data information : Transcript levels were normalized to 18S . All values shown are mean ±

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Transcript levels for Rcan1.1 and Rcan1.4 during differentiation of 3T3-L1 adipocytes, measured at 0, 1, 2, 3, 4, 6 and 24 hours following the addition MDI as well as at 3, 7 and 10 days as indicated (n=3). * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, vs 0 time point. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SDB Western blot for RCAN1.1, RCAN1.4 and tubulin (TUB) in whole cell extracts of 3T3-L1 adipocytes transfected with a control si-RNA or ones targeting either Rcan1.4 or Rcan1.1 (20 μg per lane).C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.E Transcript levels for Pgc1-α, in 3T3-L1 adipocytes transfected with the indicated si-RNAs and assayed over 10-days of differentiation (n=3). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SDRepresentative bright-field images showing lipid accumulation at 10-days differentiation of Ing-svf cells treated with the indicated siRNA. Scale bar = 50 μm.G Transcript levels for Adrenoceptor beta 3 (Adrb3), uncoupling protein 1 (Ucp1), PPARγ coactivator 1 alpha (PGC1α), and deiodinase type II (Dio2) Ing-svf cultures transfected with control siRNA (C) or one targeting Rcan1 (KD) then maintained in growth media (G), or differentiated for ten-days (D) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting (D+ISO) (n=2 in triplicate). * = p < 0.05; ** = p < 0.01, two-way ANOVA with multiple comparisons. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SDH, I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet-derived growth factor receptor alpha-positive (PDGFRα+) and lineage-negative (Lin-) stromal vascular cells isolated by FACS sorting from sWAT of WT (black bars) and KO (red bars). In H, isolated cells were maintained in growth media. In I, cultures were treated with MDI then differentiated for 10 days. * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, WT vs. KO. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


Figure 5A Representative H & E histological sections of sWAT from WT and KO mice probed with anti - UCP1 antibodies . Mice were either maintained at 24 ° C ( RT ) or housed at 6 ° C for 5 days . Scale bar = 200 μm . Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4 ° C ( n = Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple comparisonsC Transcript levels of Ucp1 , Pgc1α , Adrb3 , retinoblastoma 1 ( Rb1 ) , zinc finger protein 423 ( Zfp423 ) , and PR domain containing 16 ( Prdm16 ) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD ( n = 4 - 5 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ; unpaired student ' s t - testD Transcript levels of Pgc1α - 1b and Pgc1α - 1a isoforms in sWAT of WT and KO 8 week old females maintained at 24 ° C ( RT ) or housed at 4 ° C for 24 hours ( n = 5 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple comparisonsE . Western blot for RCAN1 in various skeletal muscle groups . Each lane was loaded with 20 μg of total protein from plantaris ( PL ) , soleus ( SOL ) , extensor digitorum longus ( EDL ) , tibialis anterior ( TA ) , red gastrocnemius ( RG ) , white gastrocnemius ( WG ) , white vastus lateralis ( WV ) , heart ventricle ( HT ) , atria , diaphragm ( Dia ) , and Tong . The lane containing brain extract was loaded with 10 μg of protein . Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1 - KO animal ( HT - KO ) . The asterisks * indicate the location of putative proteolytic fragments of RCAN1 . 1 . F Transcript levels of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old WT and KO mice housed at RT . ( n = 4 - 5 each gender ) . G Transcript levels of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old male WT and KO animals housed at RT . ( n = 5 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple comparisons F , ; unpaired student ' s t - test in G . H Representative western blot showing SLN protein in soleus muscle of 8 - 10 week old WT and KO animals housed at RT . GAPDH was used as a loading control . J Activity of the Sln - Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin ( CnA * ) and Rcan1 . Luciferase activity was normalized to beta - galactosidase activity from a co - transfected vector . ( n = 3 , assayed in duplicate ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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

Figure 5A Representative H&E histological sections of sWAT from WT and KO mice probed with anti-UCP1 antibodies. Mice were either maintained at 24°C (RT) or housed at 6°C for 5 days. Scale bar = 200 μm.Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4°C (n= Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisonsC Transcript levels of Ucp1, Pgc1α, Adrb3, retinoblastoma 1 (Rb1), zinc finger protein 423 (Zfp423), and PR domain containing 16 (Prdm16) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD (n=4-5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. ; unpaired student's t-testD Transcript levels of Pgc1α-1b and Pgc1α-1a isoforms in sWAT of WT and KO 8 week old females maintained at 24°C (RT) or housed at 4°C for 24 hours (n=5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisonsE. Western blot for RCAN1 in various skeletal muscle groups. Each lane was loaded with 20 μg of total protein from plantaris (PL), soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), red gastrocnemius (RG), white gastrocnemius (WG), white vastus lateralis (WV), heart ventricle (HT), atria, diaphragm (Dia), and Tong. The lane containing brain extract was loaded with 10 μg of protein. Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1-KO animal (HT-KO). The asterisks * indicate the location of putative proteolytic fragments of RCAN1.1.F Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old WT and KO mice housed at RT. (n=4-5 each gender). G Transcript levels of Sln in skeletal muscle (tibialis anterior ) of 8-10 week old male WT and KO animals housed at RT. (n=5).Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons F, ; unpaired student's t-test in G.H Representative western blot showing SLN protein in soleus muscle of 8-10 week old WT and KO animals housed at RT. GAPDH was used as a loading control.J Activity of the Sln-Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin (CnA*) and Rcan1. Luciferase activity was normalized to beta-galactosidase activity from a co-transfected vector. (n=3, assayed in duplicate). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


Figure 6A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA - IR , using male mice from over 100 strains from the hybrid mouse diversity panel . Regression line ( red ) , r = biweight midcorrelation , p = p - value . B , C Correlation of Rcan1 expression in gonadal adipose and liver with body weight , plasma insulin and plasma triglycerides , in a data set comparing backgrounds susceptible ( BTBR background ) or resistant ( C57BL / 6 background ) to diabetes when carrying the leptinob / ob ( ob ) mutation . Measures are reported as the ratio of the mean log10 intensity ( ml ratio ) . Regression line ( black ) , r = linear regression , p = p - value .

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

Figure 6A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA-IR, using male mice from over 100 strains from the hybrid mouse diversity panel. Regression line (red), r = biweight midcorrelation, p = p-value.B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value.


Figure 1Schematic overview of the sample collection procedure for single - cell PCR ( scPCR ) . Single IHC cytoplasms from acutely dissected organs of Corti of C57BL / 6J ( Wt ) mice after hearing onset were aspirated and processed for scPCR as depicted . ( B ' ) Expression analysis of endophilins - A1 - 3 from individually isolated IHC cytoplasms using RT - PCR from a representative experimental run . Please note that for these experiments , negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution . HPRT was used as a housekeeping gene . Immunoblotting of tissue lysates from p15 Wt ( C57BL / 6J ) , 1 - SKO , 1 / 2 - DKO and 1 / 3 - DKO revealed protein expression of endophilins - A1 and - A2 in the murine cochlea and ensured antibody specificity . Unfortunately , none of the commercially available endophilin - A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts , but rather appeared to ( also ) detect A1 . γ - adaptin was used as loading control . All antibody epitopes localize to the distinct C - terminal regions of the different endophilin - A family members . Hi , hippocampus ; Ce , cerebellum ; Co , pooled cochleae from a single individual of the indicated genotype ; Md , modioli ( micro - dissected and pooled from 10 Wt animals ) ; OC , organs of Corti ( micro - dissected and pooled from 10 Wt animals ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Schematic overview of the sample collection procedure for single-cell PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B') Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT-PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution. HPRT was used as a housekeeping gene.Immunoblotting of tissue lysates from p15 Wt (C57BL/6J), 1-SKO, 1/2-DKO and 1/3-DKO revealed protein expression of endophilins-A1 and -A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals).


Figure 2Ca2 + - current - voltage relationships evoked by incremental 15 ms step depolarizations from - 87 mV to + 58 mV revealed a ~ 25 % reduction of ICa in 1 / 3 DKO and TKO mice . ( A ' ) Due to the perinatal lethality of TKO mice , detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically - cultured organs of Corti after 7 days in vitro ( DIV ) . C57BL / 6J ( Wt ) and 1 / 3 - DKO served as controls . Please note that all recordings from cultured IHCs were performed at [ Ca2 + ] e of 10 mM to maximize IHC exocytic performance . ( A " ) Quantification and statistical analysis of individual maximum ICa amplitudes ( Imax ) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) . Ca2 + - current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1 / 3 - DKO cells . ( B ' ) Quantification and statistical analysis of the residual current at the end of the test pulse . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) . Representative ICa ( upper panel ) and Cm ( lower panel ) in response to a 50 ms depolarizing step to the potential eliciting Imax . ( C ' ) Exocytic ∆ Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2 - 100 ms for all respective genotypes and at [ Ca2 + ] e = 6 mM for a second set of recordings from wild - type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants . ( C " ) Magnification of the initial , short depolarizing steps ( 2 - 20 ms ) for clarity . Exocytic ∆ Cm of cultured endophilin - deficient IHCs appears strongly reduced . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis ) . The reduced Ca2 + - efficiency of exocytosis ( ∆ Cm / QCa ) in endophilin - deficient IHCs indicates that diminished Ca2 + - influx cannot fully account for the reduction of exocytosis . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Table S4 for detailed statistical analysis ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Ca2+-current-voltage relationships evoked by incremental 15 ms step depolarizations from -87 mV to +58 mV revealed a ~25% reduction of ICa in 1/3 DKO and TKO mice. (A') Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically-cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A") Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).Ca2+-current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B') Quantification and statistical analysis of the residual current at the end of the test pulse. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C') Exocytic ∆Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2-100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C") Magnification of the initial, short depolarizing steps (2-20 ms) for clarity. Exocytic ∆Cm of cultured endophilin-deficient IHCs appears strongly reduced. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). The reduced Ca2+-efficiency of exocytosis (∆Cm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+-influx cannot fully account for the reduction of exocytosis. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Table S4 for detailed statistical analysis).


Figure 3Ca2 + - current - voltage relationships in response to 10 ms step - depolarizations . ( A ' ) The peak of Ca2 + - influx was significantly reduced in endophilin 1 - SKO ( * * p = 0 . 0024 ) and 1 / 3 - DKO ( * * * p < 0 . 0001 ) when compared to Wt . ( Wt n = 30 / N = 20 ; 1 - SKO n = 15 / N = 9 ; 1 / 3 - DKO n = 39 / N = 20 ; Non - parametric K - W with post - hoc Dunn ' s correction ) . Quantification of CtBP2 - labeled ribbons revealed a number of approximately 14 - 15 ribbon synapses per IHC in all groups , that remained comparable across all genotypes ( Wt n = 12 / N = 9 ; 1 - SKO n = 6 / N = 4 ; 1 / 3 - DKO n = 8 / N = 6 ; one - way ANOVA , p = 0 . 717 ) . Quantification of the cross - sectional area of CaV1 . 3 immunofluorescence revealed approx . 34 % smaller clusters in 1 - SKOs and 24 % smaller clusters in 1 / 3 - DKOs . ( Wt n = 153 / N = 2 ; 1 - SKO n = 102 / N = 2 ; 1 / 3 - DKO n = 441 / N = 4 ; * * * p < 0 . 0001 ; K - W and Dunn ' s post - hoc test ) . Representative maximum projections of confocal sections from organs of Corti of p15 Wt , 1 - SKO , and 1 / 3 - DKO mice stained for CtBP2 to label synaptic ribbons . Please note that CtBP2 expression is also found in the nucleus ( nuclei are highlighted by dashed circles in the individual panels ) . Scale bars : 10 µm . 2D STED images of IHC ribbon synapses stained for CtBP2 ( magenta ) and CaV1 . 3 ( green ) . CaV1 . 3 Ca2 + - channels remain clustered at AZs of 1 - SKO and 1 / 3 - DKO IHCs . Scale bars : 1 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717).Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test).Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm.2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.


Figure 4Representative Ca2 + - currents ( upper panel ) and exocytic membrane capacitance increments ( ∆ Cm ; lower panel ) in response to a 200 ms depolarizing step to - 14 mV . Exocytic ∆ Cm ( top ) and corresponding Ca2 + - current integrals QCa ( bottom ) of Wt and endophilin - deficient IHCs in response to voltage steps from - 84 to - 14 mV of variable stimulus duration ( 5 to 200 ms ) . In 1 / 3 - DKO IHCs , our data indicate reduced sustained exocytosis to a strong depolarization ( 200 ms ; * p = 0 . 0470 , K - W with Dunn ' s post - hoc test ; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis ) . Inset : Initial ∆ Cm showed comparable RRP exocytosis in endophilin - deficient IHCs . Ca2 + - efficiency to drive exocytosis was reduced for strong depolarizations ( 200 ms ) in 1 / 3 - DKO IHCs ; * p = 0 . 0323 , K - W with Dunn ' s post - hoc test ; please also refer to Appendix Table S7 for detailed statistical analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Representative Ca2+-currents (upper panel) and exocytic membrane capacitance increments (∆Cm; lower panel) in response to a 200 ms depolarizing step to -14 mV.Exocytic ∆Cm (top) and corresponding Ca2+-current integrals QCa (bottom) of Wt and endophilin-deficient IHCs in response to voltage steps from -84 to -14 mV of variable stimulus duration (5 to 200 ms). In 1/3-DKO IHCs, our data indicate reduced sustained exocytosis to a strong depolarization (200 ms; *p = 0.0470, K-W with Dunn's post-hoc test; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis). Inset: Initial ∆Cm showed comparable RRP exocytosis in endophilin-deficient IHCs.Ca2+-efficiency to drive exocytosis was reduced for strong depolarizations (200 ms) in 1/3-DKO IHCs; *p = 0.0323, K-W with Dunn's post-hoc test; please also refer to Appendix Table S7 for detailed statistical analysis.


Figure 5Confocal maximum projections of otoferlin - immunolabeled organs of Corti from p15 Wt and 1 / 3 - DKO mice illustrated with an intensity - coded look - up table where brighter colors indicate higher intensity . Scale bars : 5 µm . Otoferlin levels were reduced by approx . 28 . 4 ± 3 . 5 % in 1 / 3 - DKO IHCs compared to Wt IHCs ( relative intensities normalized to Wt ; Wt n = 31 images ( 229 IHCs ) / N = 5 organs of Corti ( 3 animals ) ; 1 / 3 - DKO n = 20 images ( 196 IHCs ) / N = 5 organs of Corti ( 4 animals ) ; unpaired student ' s t - test ; * * * p < 0 . 001 ; box plots illustrate the median with the interquartile range , whiskers indicate 10 - 90 % of data points , the squares present the respective mean value ) . Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical ( 0 . 0 ) to basal ( 1 . 0 ) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1 / 3 - DKO and an overall reduction of otoferlin levels throughout the entire cell in 1 / 3 - DKOs . Traces indicate the mean pixel intensity ± SEM ; Wt n = 123 / N = 3 ; 1 / 3 - DKO n = 124 / N = 3 . Interaction of otoferlin and endophilin - A1 detected by co - IP in HeLa cells co - expressing GFP - otoferlin and RFP - endophilin - A1 . Otoferlin - GFP was immunoprecipitated ( IP ) by GFP - Trap beads , and blots were probed with a KO - validated anti - endophilin - A1 antibody . Exogenously overexpressed GFP - otoferlin was immunoprecipitated using GFP - Trap beads and incubated with purified endophilin - A1 ( pA1 ) . IP was then followed by immunoblotting with an anti - endophilin - A1 antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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 5Confocal maximum projections of otoferlin-immunolabeled organs of Corti from p15 Wt and 1/3-DKO mice illustrated with an intensity-coded look-up table where brighter colors indicate higher intensity. Scale bars: 5 µm.Otoferlin levels were reduced by approx. 28.4 ± 3.5 % in 1/3-DKO IHCs compared to Wt IHCs (relative intensities normalized to Wt; Wt n = 31 images (229 IHCs)/ N = 5 organs of Corti (3 animals); 1/3-DKO n = 20 images (196 IHCs)/ N = 5 organs of Corti (4 animals); unpaired student's t-test; ***p < 0.001; box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value).Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical (0.0) to basal (1.0) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1/3-DKO and an overall reduction of otoferlin levels throughout the entire cell in 1/3-DKOs. Traces indicate the mean pixel intensity ± SEM; Wt n = 123/ N = 3; 1/3-DKO n = 124/ N = 3.Interaction of otoferlin and endophilin-A1 detected by co-IP in HeLa cells co-expressing GFP-otoferlin and RFP-endophilin-A1. Otoferlin-GFP was immunoprecipitated (IP) by GFP-Trap beads, and blots were probed with a KO-validated anti-endophilin-A1 antibody.Exogenously overexpressed GFP-otoferlin was immunoprecipitated using GFP-Trap beads and incubated with purified endophilin-A1 (pA1). IP was then followed by immunoblotting with an anti-endophilin-A1 antibody.


Figure 6Endocytosis was assessed by determining the decrease in Cm during 20 s post - depolarization in perforated patch recordings from both Wt , 1 / 2 - DKO and 1 / 3 - DKO IHCs . The two kinetic components are presented as average ∆ Cm recordings after ( A ) 20 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 7 ; 1 / 3 - DKO n = 13 / N = 10 ) and ( B ) 200 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 6 ; 1 / 3 - DKO n = 14 / N = 10 ) depolarization stimuli . Average time of return to baseline obtained by fitting a linear function to the Cm - data following a 20 ms depolarization . Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses , obtained from fitting Cm - data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm - recording for 200 ms depolarizations . ( * p < 0 . 05 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ) . Average amplitude of the exponential component obtained from fits to Cm - data following 200 ms depolarization ( nexp fit = 11 for Wt , 10 for 1 / 2 - DKO and 11 for 1 / 3 - DKO ; x data points in ( E ) correspond to IHCs with no exponential component in Cm ) . correspond to statistically identified outliers . time constant of the exponential component obtained from fits to Cm - data following 200 ms

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

Figure 6Endocytosis was assessed by determining the decrease in Cm during 20 s post-depolarization in perforated patch recordings from both Wt, 1/2-DKO and 1/3-DKO IHCs. The two kinetic components are presented as average ∆Cm recordings after (A) 20 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 7; 1/3-DKO n = 13/ N = 10) and (B) 200 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 6; 1/3-DKO n = 14/ N = 10) depolarization stimuli.Average time of return to baseline obtained by fitting a linear function to the Cm-data following a 20 ms depolarization.Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses, obtained from fitting Cm-data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm- recording for 200 ms depolarizations. (*p < 0.05; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's).Average amplitude of the exponential component obtained from fits to Cm-data following 200 ms depolarization (nexp fit = 11 for Wt, 10 for 1/2-DKO and 11 for 1/3-DKO; x data points in (E) correspond to IHCs with no exponential component in Cm). correspond to statistically identified outliers.time constant of the exponential component obtained from fits to Cm-data following 200 ms depolarization


Figure 7Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center ( exemplary for a 1 / 3 - DKO specimen ) . Graphical aids indicate small vesicles , ELVs and different subpopulations of coated structures quantified in ( B - D ) . Scale bar : 500 nm . While an increased number of cytosolic vesicles can be observed at 1 - SKO AZs , reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs . RA - SVs were excluded from this quantification . Accumulations of ELVs occur in 1 / 2 - DKO and 1 / 3 - DKO IHCs , as measured by the relative cumulative ELV area per section . Increased overall number of coated structures in endophilin mutants . ( D ' ) Prominent accumulation of coated pits can be observed in 1 / 2 - DKO and 1 / 3 - DKO IHCs alongside a shift towards more coated vacuoles ( d > 70 nm ) in 1 / 2 - DKO IHCs and 1 / 3 - DKO IHCs .

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

Figure 7Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center (exemplary for a 1/3-DKO specimen). Graphical aids indicate small vesicles, ELVs and different subpopulations of coated structures quantified in (B-D). Scale bar: 500 nm.While an increased number of cytosolic vesicles can be observed at 1-SKO AZs, reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs. RA-SVs were excluded from this quantification.Accumulations of ELVs occur in 1/2-DKO and 1/3-DKO IHCs, as measured by the relative cumulative ELV area per section.Increased overall number of coated structures in endophilin mutants. (D') Prominent accumulation of coated pits can be observed in 1/2-DKO and 1/3-DKO IHCs alongside a shift towards more coated vacuoles (d > 70 nm) in 1/2-DKO IHCs and 1/3-DKO IHCs.


Figure 8Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin - A alleles . Scale bars : 200 nm . Reduced numbers of RA - SVs in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Reduced ratio of RA - SVs in the distal half of the ribbon over RA - SVs in its proximal half in 1 / 2 - DKO and 1 / 3 - DKO IHCs . K - W followed by Dunn ' s test ; * * p < 0 . 01 ; * * * p < 0 . 001 . Reduced numbers of MP - SVs ( D ) in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Significantly reduced numbers of RA - SVs in 1 / 2 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0229 ) . Significantly reduced numbers of MP - SVs in 1 / 2 - DKO and a strong trend ( p = 0 . 0511 ) towards less MP - SVs in 1 / 3 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0138 ) . Increased SV diameter in 1 / 2 - DKO , but unchanged SV diameter in 1 / 3 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0154 ) . Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1 / 2 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * * * p = 0 . 0002 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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 8Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin-A alleles. Scale bars: 200 nm.Reduced numbers of RA-SVs in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Reduced ratio of RA-SVs in the distal half of the ribbon over RA-SVs in its proximal half in 1/2-DKO and 1/3-DKO IHCs. K-W followed by Dunn's test; **p < 0.01; ***p < 0.001.Reduced numbers of MP-SVs (D) in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001.Significantly reduced numbers of RA-SVs in 1/2-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0229).Significantly reduced numbers of MP-SVs in 1/2-DKO and a strong trend (p = 0.0511) towards less MP-SVs in 1/3-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0138).Increased SV diameter in 1/2-DKO, but unchanged SV diameter in 1/3-DKOs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0154).Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1/2-DKOs (one-way ANOVA followed by Tukey's post-hoc test; ***p = 0.0002).


Figure 9Representative electron micrographs of Wt and 1 / 2 - DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity ( radius r = 1 µm from the center of the ribbon ) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K + . Scale bars : 200 nm . Significantly reduced numbers of RA - SVs in IHCs of 1 / 2 - DKO compared to Wt after stimulation , as quantified using random ultrathin sections of ribbon - occupied AZs from several IHCs per genotype . Mann - Whitney test ; * * * p < 0 . 001 . Numbers of MP - SVs were slightly reduced in stimulated Wt compared to stimulated 1 / 2 - DKOs , where SV numbers closely matched the ones of unstimulated 1 / 2 - DKO . Mann - Whitney test ; * p < 0 . 05 . Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs compared to Wt after stimulation . RA - SVs were excluded from this quantification . Student ' s t - test ; * * * p < 0 . 001 . Stimulated 1 / 2 - DKOs exhibited accumulations of ELVs in comparison to stimulated Wt . Mann - Whitney test ; * * * p < 0 . 001 . Increased overall number of coated structures in stimulated endophilin 1 / 2 - DKOs . ( F ' ) Both , coated vesicles and coated pits forming at ELVs were significantly increased in 1 / 2 - DKO after stimulation . Mann - Whitney test ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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Figure 9Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.Numbers of MP-SVs were slightly reduced in stimulated Wt compared to stimulated 1/2-DKOs, where SV numbers closely matched the ones of unstimulated 1/2-DKO. Mann-Whitney test; *p < 0.05.Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs compared to Wt after stimulation. RA-SVs were excluded from this quantification. Student's t-test; ***p < 0.001.Stimulated 1/2-DKOs exhibited accumulations of ELVs in comparison to stimulated Wt. Mann-Whitney test; ***p < 0.001.Increased overall number of coated structures in stimulated endophilin 1/2-DKOs. (F') Both, coated vesicles and coated pits forming at ELVs were significantly increased in 1/2-DKO after stimulation. Mann-Whitney test; **p < 0.01; ***p < 0.001.


Figure 1A ) Infection susceptibility ( top ) to VSVG and Spike - typed lentiviruses of a panel of lung cancer cell lines with or without ( Empty vector ) overexpression of ACE2 . ACE2 protein levels were determined by Western - blot ( bottom ) . C ) Gene - level scores obtained by robust ranking algorithm analysis of the sgRNA relative abundances between cells infected with Spike - typed lentiviruses and uninfected cells . Genes were ordered alphabetically in the x - axis and the top hits ( log10 ( enrichment - score ) < - 5 ) were colored and labelled . Note that CTSL and CTSL1 are different gene isoforms .

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

Figure 1A) Infection susceptibility (top) to VSVG and Spike-typed lentiviruses of a panel of lung cancer cell lines with or without (Empty vector) overexpression of ACE2. ACE2 protein levels were determined by Western-blot (bottom).C) Gene-level scores obtained by robust ranking algorithm analysis of the sgRNA relative abundances between cells infected with Spike-typed lentiviruses and uninfected cells. Genes were ordered alphabetically in the x-axis and the top hits (log10(enrichment-score) < -5) were colored and labelled. Note that CTSL and CTSL1 are different gene isoforms.