B . Percentage of IFNγ + / IL - 10 + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Percentage of IFNγ+/IL-10+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.


C . IL - 10 mRNA levels , determined by RNA - sequencing , in TCRβ + CD4 + cells purified from spleens of L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice ( n = 5 per group ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

C. IL-10 mRNA levels, determined by RNA-sequencing, in TCRβ+ CD4+ cells purified from spleens of L. donovani infected WT (blue) or miR-132-/- (red) mice (n=5 per group).


D . Percentage of IFNγ + WT ( blue ) or miR - 132 - / - ( red ) in vitro polarised Th1 cells ( 6 days ) in the presence or absence of phenylephrine ( PE ) , determined by intracellular cytokine staining . E . Percentage of IL10 + WT ( blue ) or miR - 132 - / - ( red ) in vitro polarised Th1 cells ( 6 days ) in the presence or absence of phenylephrine ( PE ) , determined by intracellular cytokine staining . F . Total cell counts following in vitro Th1 polarisation ( 6 days ) in the presence or absence of phenylephrine ( PE ) . For ( D - E ) , cells were purified from 3 mice per group and 6 replicates performed . Data information : statistical significance was determined with 1 - way ANOVA followed by Bonferroni ' s multiple comparison test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant .

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Percentage of IFNγ+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. E. Percentage of IL10+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. F. Total cell counts following in vitro Th1 polarisation (6 days) in the presence or absence of phenylephrine (PE). For (D-E), cells were purified from 3 mice per group and 6 replicates performed. Data information: statistical significance was determined with 1-way ANOVA followed by Bonferroni's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.


Liver LDU ( Leishman Donovan units ) at day 28 in infected WT mice treated with anti - IL - 10R antibody or isotype control antibody ( left panel , n = 5 mice per group ) , or at day 21 and day 28 from WT ( blue ) , IL - 10 + / - ( open green circles ) and IL - 10 - / - ( filled green circles ) mice ( right panel n = 3 - 6 mice per group ) Data information : Significance determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Liver LDU (Leishman Donovan units) at day 28 in infected WT mice treated with anti-IL-10R antibody or isotype control antibody (left panel, n=5 mice per group), or at day 21 and day 28 from WT (blue), IL-10+/- (open green circles) and IL-10-/- (filled green circles) mice (right panel n= 3-6 mice per group) Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT ( blue ) and miR - 132 - / - ( miR - 132 - / - ; red ) mice . Data from 4 independent infection experiments . Mean WT and miR - 132 - / - spleen parasite burdens from the 4 independent experiments shown in ( B ) . Lines link individual experiments . Splenic parasite burdens relative to WT group ( WT mean = 1 ) for each of the 4 experiments shown in ( B ) , with each data point representing individual mouse . Data information : Significance determined by unpaired t - test , and in ( C ) by paired t - test of mean values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT (blue) and miR-132-/- (miR-132-/-; red) mice. Data from 4 independent infection experiments.   Mean WT and miR-132-/- spleen parasite burdens from the 4 independent experiments shown in (B). Lines link individual experiments.   Splenic parasite burdens relative to WT group (WT mean = 1) for each of the 4 experiments shown in (B), with each data point representing individual mouse.   Data information: Significance determined by unpaired t-test, and in (C) by paired t-test of mean values. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


Spleen size expressed as % body weight for d0 ( naïve ) or day 28 L . donovani infected WT ( blue ) and miR - 132 - / - ( miR - 132 - / - ; red ) mice . Liver size expressed as % body weight for d0 ( naïve ) or day 28 L . donovani infected WT ( blue ) and miR - 132 - / - ( red ) mice . Data information : Significance determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spleen size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (miR-132-/-; red) mice.   Liver size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (red) mice.   Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair .

Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair.


Representative Western blot images of COQ9protein in kidney homogenate from Coq9 + / + ( n = 4 ) and Coq9Q95Xmice ( n = 4 ) at 3 months of age . Antibody sc - 271892 was used to map the C - terminal region of the COQ9protein and antibody ab - 104189 was used to map the internal sequence of the COQ9protein .

O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O

Representative Western blot images of COQ9protein in kidney homogenate from Coq9+/+ (n = 4) and Coq9Q95Xmice (n = 4) at 3 months of age. Antibody sc-271892 was used to map the C-terminal region of the COQ9protein and antibody ab-104189 was used to map the internal sequence of the COQ9protein.


High - resolution LC - MS / MS proteomic analysis of kidneymitochondria from Coq9 + / + ( n = 3 ) and Coq9Q95Xmice ( n = 3 ) at 3 months of age . None of the six peptides of the COQ9 protein identified in Coq9 + / + mice was detected in Coq9Q95Xmice .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O

High-resolution LC-MS/MS proteomic analysis of kidneymitochondria from Coq9+/+ (n = 3) and Coq9Q95Xmice (n = 3) at 3 months of age. None of the six peptides of the COQ9 protein identified in Coq9+/+mice was detected in Coq9Q95Xmice.


A - F CoQ9 levels in tissue homogenates from brain ( A ) , cerebellum ( B ) , heart ( C ) , kidney ( D ) , extensor ( E ) and triceps surae ( F ) of male and female Coq9 + / + and Coq9Q95Xmice at 6 and 12 months of age . Data are expressed as mean ± SD . Statistical analysis was performed on 6 - month - old Coq9 + / + mice versus 6 - month - old Coq9Q95Xmice and 12 - month - old Coq9 + / + mice versus 12 - month - old Coq9Q95Xmice . * * P < 0 . 01 ; * * * P < 0 . 001 . Student ' s t - test ( n = 8 for each group ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), extensor (E) and triceps surae (F) of male and female Coq9+/+ and Coq9Q95Xmice at 6 and 12 months of age. Data are expressed as mean ± SD. Statistical analysis was performed on 6-month-old Coq9+/+mice versus 6-month-old Coq9Q95Xmice and 12-month-old Coq9+/+mice versus 12-month-old Coq9Q95Xmice. **P < 0.01; ***P < 0.001. Student's t-test (n = 8 for each group).


A - F Residual CoQ9 levels in tissue homogenates from brain ( A ) , cerebellum ( B ) , heart ( C ) , kidney ( D ) , liver ( E ) and skeletal muscle ( F ) of Coq9 + / + , Coq9Q95X and Coq9R239X mice at 1 , 3 and 5 months of age . Data are expressed as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; # # P < 0 . 01 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice ( one - way ANOVA with a Tukey ' s post hoc test ; n = 8 for each group ; numbers above columns indicate P - values of the one - way ANOVA test ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F Residual CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), liver (E) and skeletal muscle (F) of Coq9+/+, Coq9Q95X and Coq9R239X mice at 1, 3 and 5 months of age. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice (one-way ANOVA with a Tukey's post hoc test; n = 8 for each group; numbers above columns indicate P-values of the one-way ANOVA test).


A - E mRNA expression levels of Coq9 ( A ) , Coq7 ( B ) , Adck3 ( C ) , Coq5 ( D ) and Coq6 ( E ) on cerebrum of Coq9 + / + , Coq9Q95X and Coq9Q95X mice at 3 months of age . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice . F - J mRNA expression levels of Coq9 ( F ) , Coq7 ( G ) , Adck3 ( H ) , Coq5 ( I ) and Coq6 ( J ) on kidney of Coq9 + / + , Coq9Q95X and Coq9Q95Xmice at 3 months of age . * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice . K - O mRNA expression levels of Coq9 ( K ) , Coq7 ( L ) , Adck3 ( M ) , Coq5 ( N ) and Coq6 ( O ) on triceps surae of Coq9 + / + , Coq9Q95X and Coq9Q95Xmice at 3 months of age . * P < 0 . 05 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; Coq9Q95X versus Coq9R239Xmice .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O

A-E mRNA expression levels of Coq9 (A), Coq7 (B), Adck3 (C), Coq5 (D) and Coq6 (E) on cerebrum of Coq9+/+, Coq9Q95X and Coq9Q95X mice at 3 months of age. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.F-J mRNA expression levels of Coq9 (F), Coq7 (G), Adck3 (H), Coq5 (I) and Coq6 (J) on kidney of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.K-O mRNA expression levels of Coq9 (K), Coq7 (L), Adck3 (M), Coq5 (N) and Coq6 (O) on triceps surae of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. *P < 0.05; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice.


A - D Representative Western blot and quantitation of Western blot bands of COQ7 ( A ) , ADCK3 ( B ) , COQ5 ( C ) and COQ6 ( D ) , and VDAC1 as a loading control in the kidneys of 3 - month - old mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239X mice versus Coq9 + / + mice . # # P < 0 . 01 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239X mice . One - way ANOVA with a Tukey ' s post hoc test . E - H Representative Western blot and quantitation of Western blot bands of COQ7 ( E ) , ADCK3 ( F ) , COQ5 ( G ) and COQ6 ( H ) , and VDAC1 as a loading control in skeletal muscle of 3 - month - old mice . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239X mice versus Coq9 + / + mice . # P < 0 . 05 ; # # P < 0 . 01 ; Coq9Q95X versus Coq9R239X mice . Data information : All values are presented as mean ± SD . One - way ANOVA with a Tukey ' s post hoc test . Numbers above columns indicate P - values of the one - way ANOVA test . Coq9 + / + mice n = 4 ; Coq9Q95X and Coq9R239Xmice n = 5 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O

A-D Representative Western blot and quantitation of Western blot bands of COQ7 (A), ADCK3 (B), COQ5 (C) and COQ6 (D), and VDAC1 as a loading control in the kidneys of 3-month-old mice. *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239X mice versus Coq9+/+mice. ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239X mice. One-way ANOVA with a Tukey's post hoc test.E-H Representative Western blot and quantitation of Western blot bands of COQ7 (E), ADCK3 (F), COQ5 (G) and COQ6 (H), and VDAC1 as a loading control in skeletal muscle of 3-month-old mice. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239X mice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; Coq9Q95X versus Coq9R239X mice.Data information: All values are presented as mean ± SD. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test. Coq9+/+mice n = 4; Coq9Q95X and Coq9R239Xmice n = 5.


A - C Mitochondrial CoQ9 levels from cerebrum ( A ) , kidney ( B ) and skeletal muscle ( C ) of Coq9 + / + and Coq9Q95X males and females . n = 8 for each group .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O

A-C Mitochondrial CoQ9 levels from cerebrum (A), kidney (B) and skeletal muscle (C) of Coq9+/+ and Coq9Q95X males and females. n = 8 for each group.


D - F CI + CIII activity in cerebrum ( D ) , kidney ( E ) and skeletal muscle ( F ) of male and female Coq9 + / + and Coq9Q95X mice . n = 6 for each group . G - I CII + CIII activity in cerebrum ( G ) , kidney ( H ) and skeletal muscle ( I ) of male and female Coq9 + / + and Coq9Q95X mice . n = 6 for each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O

D-F CI+CIII activity in cerebrum (D), kidney (E) and skeletal muscle (F) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.G-I CII+CIII activity in cerebrum (G), kidney (H) and skeletal muscle (I) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.


J - L Blue - native gel electrophoresis ( BNGE ) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9 + / + ( n = 3 ) and Coq9Q95X mice ( n = 4 ) at 3 months of age .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

J-L Blue-native gel electrophoresis (BNGE) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9+/+ (n = 3) and Coq9Q95X mice (n = 4) at 3 months of age.


A - D Measurement of phosphorylating respiration ( represented as State 3o , in the presence of ADP and substrates ) in kidney ( A ) and skeletal muscle ( C ) from male and female Coq9 + / + , Coq9Q95X and Coq9R239X mice at 3 months of age . Representative O2 consumption graphic in kidney ( B ) and skeletal muscle ( D ) from female Coq9 + / + , Coq9Q95X and Coq9R239X mice . Data information : All values are presented as mean ± SD . ( A , C ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; Coq9Q95X versus Coq9R239Xmice . One - way ANOVA with a Tukey ' s post hoc test . Numbers above columns indicate P - values of the one - way ANOVA test ( n = 3 for each group ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D Measurement of phosphorylating respiration (represented as State 3o, in the presence of ADP and substrates) in kidney (A) and skeletal muscle (C) from male and female Coq9+/+, Coq9Q95X and Coq9R239X mice at 3 months of age. Representative O2 consumption graphic in kidney (B) and skeletal muscle (D) from female Coq9+/+, Coq9Q95X and Coq9R239X mice.Data information: All values are presented as mean ± SD. (A, C) *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test (n = 3 for each group).


A - H Complex II ( SDH ) and complex IV ( COX ) histochemistry of triceps surae showing a decreased stain in 18 - month - old Coq9Q95X female mice ( D , H ) in contrast to normal SDH and COX activity in 6 - and 18 - month - old Coq9 + / + ( A , C , E , G ) , as well as 6 - month - old Coq9Q95X female mice ( B , F ) . I - L Gomori trichrome stain ( TGM ) of triceps surae showed no differences between 6 - and 18 - month - old Coq9 + / + and Coq9Q95X female mice . M - P Hematoxylin and eosin ( H & amp ; amp ; E ) stains of triceps surae did not reveal any structural abnormality . Data information : Scale bars : 100 μm . n = 3 for each group . Complex IV , cytochrome c oxidase ( COX ) ; complex II , succinate dehydrogenase ( SDH ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-H Complex II (SDH) and complex IV (COX) histochemistry of triceps surae showing a decreased stain in 18-month-old Coq9Q95X female mice (D, H) in contrast to normal SDH and COX activity in 6- and 18-month-old Coq9+/+ (A, C, E, G), as well as 6-month-old Coq9Q95X female mice (B, F).I-L Gomori trichrome stain (TGM) of triceps surae showed no differences between 6- and 18-month-old Coq9+/+ and Coq9Q95X female mice.M-P Hematoxylin and eosin (H&amp;E) stains of triceps surae did not reveal any structural abnormality.Data information: Scale bars: 100 μm. n = 3 for each group. Complex IV, cytochrome c oxidase (COX); complex II, succinate dehydrogenase (SDH).


A - C Voluntary wheel running test . Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age ( B , C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

A-C Voluntary wheel running test. Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age (B, C).


D Hanging wire test . Coq9Q95X female mice obtained less reaches score in the ' fall and reaches ' method .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

D Hanging wire test. Coq9Q95X female mice obtained less reaches score in the 'fall and reaches' method.


E Open - field test . Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

E Open-field test. Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age.


F Grip test : Muscle strength was not affected in Coq9Q95X mice at 6 months of age .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

F Grip test: Muscle strength was not affected in Coq9Q95X mice at 6 months of age.


A , B KidneyCoQ9 levels in Coq9 + / + , Coq9Q95X and Coq9R239Xmice treated with 2 , 4 - diHB ( + 2 , 4 - diHB ) compared with the non - treated littermate ( vehicle ) . Statistical analysis was performed on + 2 , 4 - diHBCoq9 + / + , Coq9Q95X and Coq9R239Xmice versus vehicle Coq9 + / + , Coq9Q95X and Coq9R239Xmice , respectively ( n = 3 for each group ) .

O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

A, B KidneyCoQ9 levels in Coq9+/+, Coq9Q95X and Coq9R239Xmice treated with 2,4-diHB (+2,4-diHB) compared with the non-treated littermate (vehicle). Statistical analysis was performed on +2,4-diHBCoq9+/+, Coq9Q95X and Coq9R239Xmice versus vehicle Coq9+/+, Coq9Q95X and Coq9R239Xmice, respectively (n = 3 for each group).


C , D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2 , 4 - diHB ( + 2 , 4 - diHB , blue bars ) compared with the non - treated controls ( vehicle , yellow bars ) . Statistical analysis was performed on + 2 , 4 - diHBCOQ9R244X versus vehicle COQ9R244X ( n = 4 for each group ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O

C, D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2,4-diHB (+2,4-diHB, blue bars) compared with the non-treated controls (vehicle, yellow bars). Statistical analysis was performed on +2,4-diHBCOQ9R244X versus vehicle COQ9R244X (n = 4 for each group).


( a ) Immunoblot for the indicated Cx of homogenates ( Hom ) and autophagic vacuoles ( AV ) isolated from fed or 6 h starved ( Stv ) mice . Quantification of enrichment is shown in Supplementary Fig . 1a .

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

(a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a.


( b ) Immunofluorescence for LC3 and Atg16 in normal rat kidney ( NRK ) cells expressing GFP - Cx43 maintained in the presence or absence of serum for 4 h . Single channels are shown in reverse black and white , and magnification of single and merged images are shown in colour . Arrows : co - localization of Atg16 / Cx43 ( yellow ) or Atg16 / Cx43 / LC3 ( white ) . Quantification is shown in Supplementary Fig . 1c .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

(b) Immunofluorescence for LC3 and Atg16 in normal rat kidney (NRK) cells expressing GFP-Cx43 maintained in the presence or absence of serum for 4 h. Single channels are shown in reverse black and white, and magnification of single and merged images are shown in colour. Arrows: co-localization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c.


( c ) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum - supplemented media without additions ( None ) or treated with chloroquine ( CQ ) for 4 h . Full fields are shown in Supplementary Fig . 1d .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4 h. Full fields are shown in Supplementary Fig. 1d.


( d ) Maximum projection confocal microscopy 3D reconstruction ( left ) followed by a single - plane time - lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and sfGFP - ATG16 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O

(d) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single-plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and sfGFP-ATG16.


( e ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP - Cx43 but knocked down for Eps15 . Single channels are shown in reverse black and white , and merged images in colour . Inset shows higher magnification . Bottom : Quantification of co - localization between Atg16 and Cx43 ( n = 3 wells , 4 independent experiments , > 20 cells per experiment ) . Values are mean + s . e . m . * P 0 . 05 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white, and merged images in colour. Inset shows higher magnification. Bottom: Quantification of co-localization between Atg16 and Cx43 (n = 3 wells, 4 independent experiments, >20 cells per experiment). Values are mean + s.e.m. * P 0.05.


( f , g ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence ( f , g ) or absence of serum ( 4 h ; g ) . I : input ; FT : flow through .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f,g) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence (f,g) or absence of serum (4 h; g). I: input; FT: flow through.


( h ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of endogenous Cx43 from untreated WT MEFs , on incubation with 3 - methyladenine ( 3MA ) or in MEFs from Atg5 - null mice ( Atg5 − / − ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O

(h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from untreated WT MEFs, on incubation with 3-methyladenine (3MA) or in MEFs from Atg5-null mice (Atg5−/−).


( i ) Immunoblot for Flag and Cx43 of immunoprecipitates ( IP ) of endogenous Cx43 in WT MEFs transfected with the indicated Flag - tagged constructs of truncated Atg16 .

O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O

(i) Immunoblot for Flag and Cx43 of immunoprecipitates (IP) of endogenous Cx43 in WT MEFs transfected with the indicated Flag-tagged constructs of truncated Atg16.


( a ) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43 - null mice ( Cx43 − / − ) grown in serum - supplemented media . Reverse black and white channels ( top ) and colour images ( bottom ) . Right : Average number of LC3 and GABARAP puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(a) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43-null mice (Cx43−/−) grown in serum-supplemented media. Reverse black and white channels (top) and colour images (bottom). Right: Average number of LC3 and GABARAP puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( b ) Electron micrographs of WT and Cx43 − / − MOBs grown in serum - supplemented media . Yellow arrows : autophagic vacuoles . Insets : autophagic vacuoles ( AV ) at higher magnification . Right : Percentage of cytoplasm area occupied by AV ( top ) and number of AV per area ( bottom ; n = 3 mice , > 10 micrographs per mice ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Electron micrographs of WT and Cx43−/− MOBs grown in serum-supplemented media. Yellow arrows: autophagic vacuoles. Insets: autophagic vacuoles (AV) at higher magnification. Right: Percentage of cytoplasm area occupied by AV (top) and number of AV per area (bottom; n = 3 mice, >10 micrographs per mice).


( c ) Immunoblot for LC3 of the same cells grown in serum - supplemented media untreated or treated with lysosomal protease inhibitors ( PI ) for 2 or 4 h . Top : representative immunoblot . GAPDH is shown as loading control . Bottom : LC3 flux ( left ) and increase in LC3 - II during lysosomal inhibition ( right ; n = 3 independent experiments ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(c) Immunoblot for LC3 of the same cells grown in serum-supplemented media untreated or treated with lysosomal protease inhibitors (PI) for 2 or 4 h. Top: representative immunoblot. GAPDH is shown as loading control. Bottom: LC3 flux (left) and increase in LC3-II during lysosomal inhibition (right; n = 3 independent experiments).


( d ) Representative images of the same cells transfected with the mCherry - GFP - LC3 tandem reporter and grown in serum - supplemented media . Single - channel images ( inverted black and white ) and merged images ( colour ) . Bottom : Number of vesicles positive for GFP and mCherry ( autophagosomes ; APG ) and only for mCherry ( autophagolysosomes ; APL ; n = 4 wells , 4 independent experiments , > 30 cells per experiment ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Representative images of the same cells transfected with the mCherry-GFP-LC3 tandem reporter and grown in serum-supplemented media. Single-channel images (inverted black and white) and merged images (colour). Bottom: Number of vesicles positive for GFP and mCherry (autophagosomes; APG) and only for mCherry (autophagolysosomes; APL; n = 4 wells, 4 independent experiments, >30 cells per experiment).


( e ) Electron micrographs of livers from WT and Cx43 + / − mice . Arrows indicate AV . Right : Percentage of cytosolic area occupied by AVs ( n = 3 mice , > 10 micrographs per mice ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Electron micrographs of livers from WT and Cx43+/− mice. Arrows indicate AV. Right: Percentage of cytosolic area occupied by AVs (n = 3 mice, >10 micrographs per mice).


( f ) Immunofluorescence for LC3 in Cx43 − / − MOBs expressing GFP - tagged full - length Cx43 . Single channels ( inverted black and white ) and merged channels ( colour ) . Asterisks : transfected ( green ) and untransfected ( red ) cells . ( g ) LC3 - positive puncta per cell in the indicated cells ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . All values are mean + s . e . m . and differences from WT were significant for * P 0 . 01 . Nuclei are highlighted with DAPI . Scale bars : 5 μm in fluorescence images ; 1 μm and 0 . 1 μm in top panels and insets of b and e . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Immunofluorescence for LC3 in Cx43−/− MOBs expressing GFP-tagged full-length Cx43. Single channels (inverted black and white) and merged channels (colour). Asterisks: transfected (green) and untransfected (red) cells. (g) LC3-positive puncta per cell in the indicated cells (n = 3 wells, 3 independent experiments, >50 cells per experiment). All values are mean + s.e.m. and differences from WT were significant for *P 0.01. Nuclei are highlighted with DAPI. Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of b and e. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblot for LC3 in MOBs from WT or Cx43 - null mice ( Cx43 − / − ) treated with vehicle or with 18α glycyrrhetinic acid ( 18α GA ) . Glyceraldehyde - 3 - dehydrogenae ( GPD ) is shown as the loading control . Levels of LC3 - II ( left ) and LC3 - II flux ( right ; n = 3 independent experiments ) .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

(a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as the loading control. Levels of LC3-II (left) and LC3-II flux (right; n = 3 independent experiments).


( b ) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid . Where indicated , chloroquine ( CQ ) was added to block lysosomal degradation . Bottom : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) .

O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(b) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).


( c ) Immunofluorescence for LC3 in WT or Cx43 − / − MOBs treated with the indicated kinase inhibitors for 2 h . Insets : Higher magnification images in inverted black and white . ( d ) Endogenous LC3 puncta in the same cells shown in c ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) . * , differences from WT ; § , differences from untreated .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Immunofluorescence for LC3 in WT or Cx43−/− MOBs treated with the indicated kinase inhibitors for 2 h. Insets: Higher magnification images in inverted black and white. (d) Endogenous LC3 puncta in the same cells shown in c (n = 3 wells, 3 independent experiments, >50 cells per experiment). *, differences from WT; §, differences from untreated.


( e ) LC3 puncta in Cx43 − / − MOBs transfected with constructs coding for different forms of Cx43 ( full length , CT , ΔCT258 or ΔCT245 ) tagged as indicated in the scheme . Left : Representative images . Merged fields ( left ) and LC3 channel in reverse black and white ( right ) are shown ( images of GFP - Cx43 are shown in Fig . 2f ) . Right : Quantification of the average number of LC3 puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(e) LC3 puncta in Cx43−/− MOBs transfected with constructs coding for different forms of Cx43 (full length, CT, ΔCT258 or ΔCT245) tagged as indicated in the scheme. Left: Representative images. Merged fields (left) and LC3 channel in reverse black and white (right) are shown (images of GFP-Cx43 are shown in Fig. 2f). Right: Quantification of the average number of LC3 puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( f , g ) Immunoblot for the indicated proteins of the immunoprecipitates ( IP ) of Atg16 from MEFs transfected with GFP - tagged full - length ( FL - ) Cx43 or Flag - tagged C terminus ( CT - ) Cx43 ( f ) or ΔCT258 - Cx43 ( g ) I : input ; FT : flow through . GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control . All values are mean + s . e . m . Differences from the control were significant for ( * , § ) P 0 . 01 and ( * * , § § ) P 0 . 001 . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f,g) Immunoblot for the indicated proteins of the immunoprecipitates (IP) of Atg16 from MEFs transfected with GFP-tagged full-length (FL-) Cx43 or Flag-tagged C terminus (CT-) Cx43 (f) or ΔCT258-Cx43 (g) I: input; FT: flow through. GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control. All values are mean + s.e.m. Differences from the control were significant for (*, §)P 0.01 and (**, §§)P 0.001. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblots for the indicated proteins in MEFs treated with tamoxifen ( Tx ) or lindane ( Lind ) in the absence or presence of lysosomal protease inhibitors ( PI ) . Right : Changes in LC3 - II levels relative to those detected in untreated cells ( n = 4 independent experiments ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(a) Immunoblots for the indicated proteins in MEFs treated with tamoxifen (Tx) or lindane (Lind) in the absence or presence of lysosomal protease inhibitors (PI). Right: Changes in LC3-II levels relative to those detected in untreated cells (n = 4 independent experiments).


( b ) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine ( CQ ) for 3 h . Right : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . ( c ) Time course of changes in the average LC3 - positive puncta per cell after addition of lindane to control NRK cells .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O

(b) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine (CQ) for 3 h. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Time course of changes in the average LC3-positive puncta per cell after addition of lindane to control NRK cells.


( d ) Fluorescence images of WT or Cx43 − / − MOBs transfected with mCherry - GFP - LC3 and incubated or not with tamoxifen or lindane . Bottom : Quantification of autophagolysosomes ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(d) Fluorescence images of WT or Cx43−/− MOBs transfected with mCherry-GFP-LC3 and incubated or not with tamoxifen or lindane. Bottom: Quantification of autophagolysosomes (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( e ) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds . Representative images as merged channels are shown . Right : quantification of the average of cytosolic Atg16 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 30 cells per experiment ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(e) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds. Representative images as merged channels are shown. Right: quantification of the average of cytosolic Atg16-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).


( f ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP - Cx43 treated with lindane and untreated . Single channel images in inverse black and white and merged colour images are shown . Outlined areas are shown at higher magnification . Arrows : double ( yellow ) and triple ( white ) co - localization .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 treated with lindane and untreated. Single channel images in inverse black and white and merged colour images are shown. Outlined areas are shown at higher magnification. Arrows: double (yellow) and triple (white) co-localization.


( g , h ) Immunostaining for Atg16 in Cx43 knocked down ( KD ) ( g ) or knocked out ( − / − ) ( h ) cells . Right : quantification of the average of cytosolic Atg16 positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(g,h) Immunostaining for Atg16 in Cx43 knocked down (KD) (g) or knocked out (−/−) (h) cells. Right: quantification of the average of cytosolic Atg16 positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( i ) Immunoblot for Atg16 and Atg14 in immunoprecipitates ( IP ) of endogenous Cx43 from WT MEFs maintained in the absence of serum ( 4 h ) or incubated in the presence of tamoxifen or lindane . I : input ; FT : flow through . All values are mean + s . e . m and differences from untreated cells are significant for * P 0 . 01 . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Immunoblot for Atg16 and Atg14 in immunoprecipitates (IP) of endogenous Cx43 from WT MEFs maintained in the absence of serum (4 h) or incubated in the presence of tamoxifen or lindane. I: input; FT: flow through. All values are mean + s.e.m and differences from untreated cells are significant for *P 0.01. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a - c ) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP - Cx43 maintained in the presence of serum ( a ) , absence of serum ( b ) or treated with lindane ( c ) . Single black and white inverted channels , merged channels and higher magnification insets are shown .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(a-c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP-Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c). Single black and white inverted channels, merged channels and higher magnification insets are shown.


( d ) Immunoblots for indicated proteins in isolated fractions from starved mouse liver . Hom , homogenate ; LE , late endosomes ; Cyt , cytosol ; APG , autophagosomes ; APL , autophagolysosomes .

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

(d) Immunoblots for indicated proteins in isolated fractions from starved mouse liver. Hom, homogenate; LE, late endosomes; Cyt, cytosol; APG, autophagosomes; APL, autophagolysosomes.


( e ) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice .

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

(e) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice.


( f - h ) Fluorescence images of NRK cells expressing GFP - Cx43 maintained in the presence of serum ( f ) , absence of serum ( g ) or treated with lindane ( h ) and incubated with Alexa594 - transferrin for 15 min before fixation . Top : single and merged channels and outlined area at higher magnification . Bottom : 3D reconstructions and higher magnification details of the outlined areas . Nuclei are highlighted with DAPI . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f-h) Fluorescence images of NRK cells expressing GFP-Cx43 maintained in the presence of serum (f), absence of serum (g) or treated with lindane (h) and incubated with Alexa594-transferrin for 15 min before fixation. Top: single and merged channels and outlined area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the outlined areas. Nuclei are highlighted with DAPI. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblot for the indicated proteins in homogenates ( Hom ) and purified PM from fed ( F ) and 24 h starved mice ( Stv ) . Starvation - induced changes in the PM content relative to fed levels were as follows : Vps15 ( 1 . 2 ± 0 . 2 ) , Beclin - 1 ( 0 . 7 ± 0 . 5 ) , Vps34 ( 0 . 9 ± 0 . 1 ) , Atg16 ( 1 . 1 ± 0 . 3 ) , Atg14 ( 2 . 5 ± 0 . 2 ) and only Atg14 were significant for P 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

(a) Immunoblot for the indicated proteins in homogenates (Hom) and purified PM from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2 ± 0.2), Beclin-1 (0.7 ± 0.5), Vps34 (0.9 ± 0.1), Atg16 (1.1 ± 0.3), Atg14 (2.5 ± 0.2) and only Atg14 were significant for P 0.01.


( b ) Immunoblot of Cx43immunoprecipitates ( IP ) in MEFs maintained in the presence or absence of serum . I , input ; FT , flow through .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(b) Immunoblot of Cx43immunoprecipitates (IP) in MEFs maintained in the presence or absence of serum. I, input; FT, flow through.


( c ) Immunoblot of Cx43 IP in MEFs from WT or Atg5 − / − mice grown in serum - supplemented media .

O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(c) Immunoblot of Cx43 IP in MEFs from WT or Atg5−/− mice grown in serum-supplemented media.


( d ) Immunoblot of Cx43 IP in WT or Atg16 knockdown ( KD ) MEFs maintained in the presence / absence of serum for 4 h .

O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(d) Immunoblot of Cx43 IP in WT or Atg16 knockdown (KD) MEFs maintained in the presence/absence of serum for 4 h.


( e ) Co - staining for Atg14 and Cx43 in NRK cells maintained in the presence / absence of serum . Merged channels are shown and single channels and broad - field images are shown in Supplementary Fig . 5d . Insets : higher magnification .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Co-staining for Atg14 and Cx43 in NRK cells maintained in the presence/absence of serum. Merged channels are shown and single channels and broad-field images are shown in Supplementary Fig. 5d. Insets: higher magnification.


( f ) Maximum - intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and EGFP - ATG14 followed by a single - plane time - lapse acquisition of the outlined area ; cells were incubated in serum - deprived medium for 1 h before imaging .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Maximum-intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and EGFP-ATG14 followed by a single-plane time-lapse acquisition of the outlined area; cells were incubated in serum-deprived medium for 1 h before imaging.


( g ) Immunoblot of Atg14 IP in WT or Atg5 − / − MEFs maintained in the presence of serum .

O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(g) Immunoblot of Atg14 IP in WT or Atg5−/− MEFs maintained in the presence of serum.


( h ) Immunoblot in total homogenates and PM purified from WT or Cx43 − / − MOBs .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

(h) Immunoblot in total homogenates and PM purified from WT or Cx43−/− MOBs.


( i ) Immunofluorescence for e - cadherin and Vps34 in NRK cells WT or knocked down ( KD ) for Cx43 . Single channel and merge ( left ) and z - stack surface reconstruction and higher magnification of the outlined region ( right ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Immunofluorescence for e-cadherin and Vps34 in NRK cells WT or knocked down (KD) for Cx43. Single channel and merge (left) and z-stack surface reconstruction and higher magnification of the outlined region (right).


( j ) Immunostaining for Cx43 and e - cadherin in NRK cells expressing a tandem FYVE domain ( 2xFYVE ) tagged to GFP and maintained in serum - free media for 4 h . Single channels ( inverted black and white ) and the merged image of the outlined area ( colour ) . Inset : higher magnification image . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(j) Immunostaining for Cx43 and e-cadherin in NRK cells expressing a tandem FYVE domain (2xFYVE) tagged to GFP and maintained in serum-free media for 4 h. Single channels (inverted black and white) and the merged image of the outlined area (colour). Inset: higher magnification image. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Co - immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum ( top ) . Single and merged channels of the 3D reconstruction of the outlined regions are shown ( bottom ) . Nuclei are highlighted with DAPI .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the outlined regions are shown (bottom). Nuclei are highlighted with DAPI.


( b , c ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of Cx43 in NRK cells maintained in the presence or absence of serum ( b ) or treated or not with 3 - methyladenine ( c ) . Atg5 is shown as the negative control for immunoprecipitation .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

(b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation.


( d ) Co - staining for Atg9 and e - cadherin in control NRK cells and those knocked down for Cx43 . Single and merged channels at higher magnification areas are shown .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(d) Co-staining for Atg9 and e-cadherin in control NRK cells and those knocked down for Cx43. Single and merged channels at higher magnification areas are shown.


( e ) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O

(e) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15.


( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg14 ( f ) maintained in the presence or absence of serum .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg14 (f) maintained in the presence or absence of serum.


( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg9 ( g ) maintained in the presence or absence of serum . E - cadherin staining ( red ) is shown to highlight PM in g and h .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg9 (g) maintained in the presence or absence of serum. E-cadherin staining (red) is shown to highlight PM in g and h.


( h ) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14 , treated ( or not ) with tamoxifen or lindane ( left ) . E - cadherin staining ( red ) is shown to highlight PM in g and h . Full fields are shown in Supplementary Fig . 6h . Quantification of intracytoplasmic Cx43 - positives vesicles ( right ; n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . Values are mean + s . e . m and significant for * P 0 . 01 and * * P 0 . 001 using analysis of variance + Bonferroni test . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblot for the indicated proteins in control MEFs or those knocked down ( KD ) for the three different Cx .

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

(a) Immunoblot for the indicated proteins in control MEFs or those knocked down (KD) for the three different Cx.


( b ) Immunofluorescence of LC3 in control MEFs ( Ctr ) or those knocked down ( KD ) for three different Cx maintained in the presence or absence of serum . Left : representative fields and higher magnification insets . Nuclei are highlighted with DAPI . Right : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(b) Immunofluorescence of LC3 in control MEFs (Ctr) or those knocked down (KD) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( c ) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated ( − ) or treated with inhibitors of lysosomal proteases ( PI ) for the indicated times . GAPDH is shown as the loading control .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (−) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as the loading control.


( d ) Representative images of control MEFs or those knocked down ( KD ) for the indicated proteins transfected with the mCherry - GFP - LC3 tandem reporter . Right : quantification of the average number per cell of puncta positive for GFP and mCherry ( autophagosomes ; APG ) and those positive only for mCherry ( autophagolysosomes ; APL ; n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Representative images of control MEFs or those knocked down (KD) for the indicated proteins transfected with the mCherry-GFP-LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL; n = 3 wells, 3 independent experiments, >50 cells per experiment).


( e ) Electron micrographs of control MEFs or those knocked down for the indicated Cx . Insets show autophagic vesicles . Bottom : Quantification of the average number of AV ( left ) and percentage of cytosolic area ( right ) occupied by AVs ( n = 4 wells , 4 independent experiments , > 10 micrographs per well ) .

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

(e) Electron micrographs of control MEFs or those knocked down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n = 4 wells, 4 independent experiments, >10 micrographs per well).


( f ) Immunoblot for indicated proteins in immunoprecipitates ( IP ) for Cx26 or Cx32 ( left ) or Atg16 ( right ) from liver homogenates . Inp : input , FT : flow through . BIP is shown as the negative control for immunoprecipitation . All values are mean + s . e . m and significant for * P 0 . 01 ( analysis of variance + Bonferroni test was used in b ) . Scale bars : 5 μm in fluorescence images ; 1 μm and 0 . 1 μm in top panels and insets of e . Uncropped images of blots are shown in Supplementary Fig . 9 .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Immunoblot for indicated proteins in immunoprecipitates (IP) for Cx26 or Cx32 (left) or Atg16 (right) from liver homogenates. Inp: input, FT: flow through. BIP is shown as the negative control for immunoprecipitation. All values are mean + s.e.m and significant for *P 0.01 (analysis of variance + Bonferroni test was used in b). Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of e. Uncropped images of blots are shown in Supplementary Fig. 9.


A . Localization of TRAPP complex components after sodium arsenite ( SA ) treatment in HeLa cells treated with 300 µM SA for 30 min . Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 ( to label SGs ) at steady state and after SA treatment ( top two rows ) . Other panels show localization of GFP - TRAPPC3 and endogenous TRAPPC1 to eIF3 - labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP - labeled SGs . DAPI ( blue ) . Scale bar , 10 µm .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

A. Localization of TRAPP complex components after sodium arsenite (SA) treatment in HeLa cells treated with 300 µM SA for 30 min. Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 (to label SGs) at steady state and after SA treatment (top two rows). Other panels show localization of GFP-TRAPPC3 and endogenous TRAPPC1 to eIF3-labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP-labeled SGs. DAPI (blue). Scale bar, 10 µm.


B . Representative images of a time course analysis of TRAPPC2 redistribution to SGs . Cells were treated The graph ( B ' ) shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 ( mean fluorescence intensity ) in SG puncta and cytosolic TRAPPC2 . Mean ± s . e . m . n = 50 cells per experiment , N = 3 . Scale bar , 10 µm .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative images of a time course analysis of TRAPPC2 redistribution to SGs. Cells were treated The graph (B') shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 (mean fluorescence intensity) in SG puncta and cytosolic TRAPPC2. Mean ± s.e.m. n = 50 cells per experiment, N = 3. Scale bar, 10 µm.


C . Analysis of TRAPPC2 ( green ) , TRAPPC8 ( blue ) and TRAPPC9 ( orange ) localization at SGs upon TRAPPC2 , TRAPPC4 TRAPPC8 and TRAPPC9 depletion . Mean ± s . e . m . n > 100 . * p < 0 . 05 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Analysis of TRAPPC2 (green), TRAPPC8 (blue) and TRAPPC9 (orange) localization at SGs upon TRAPPC2, TRAPPC4 TRAPPC8 and TRAPPC9 depletion. Mean ± s.e.m. n>100. *p<0.05; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


E . Analysis of TRAPPC2 localization at SGs upon CLIC1 , CRYAB and ENO1 depletion . Mean ± s . e . m . n > 100 . * * * * p < 0 . 0001 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Analysis of TRAPPC2 localization at SGs upon CLIC1, CRYAB and ENO1 depletion. Mean ± s.e.m. n>100. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


A . HeLa cells , untreated or treated with SA , were fixed and visualized by fluorescence microscopy using anti - Sec24C Ab , anti - eIF3 Ab , and DAPI ( blue ) . Data information : Scale bars , 10 µm .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

A. HeLa cells, untreated or treated with SA, were fixed and visualized by fluorescence microscopy using anti-Sec24C Ab, anti-eIF3 Ab, and DAPI (blue). Data information: Scale bars, 10 µm.


B . Quantification of Sec24C redistribution to SGs over time after SA treatment ( the ratio between Sec24C ( mean fluorescence intensity ) in SG puncta and cytosolic Sec24C ) . Mean ± s . e . m . n = 50 cells per experiment , N = 3 .

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

B. Quantification of Sec24C redistribution to SGs over time after SA treatment (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± s.e.m. n = 50 cells per experiment, N = 3.


C . Quantification of residual Sec24C in ERES after SA treatment . The data are expressed as percentage of steady state values ( CTRL ) . Mean ± s . e . m . n = 100 cells per experiment , N = 3 .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of residual Sec24C in ERES after SA treatment. The data are expressed as percentage of steady state values (CTRL). Mean ± s.e.m. n = 100 cells per experiment, N = 3.


D . Representative images of TRAPPC2 localization in Sec23AB - KD and Sec24ABCD - KD cells treated with SA . Cells were fixed and visualized by fluorescence microscopy using anti - TRAPPC2 Ab , anti - G3BP Ab ( to label SGs ) and DAPI ( blue ) . Data information : Scale bars , 10 µm .

O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

D. Representative images of TRAPPC2 localization in Sec23AB-KD and Sec24ABCD-KD cells treated with SA. Cells were fixed and visualized by fluorescence microscopy using anti-TRAPPC2 Ab, anti-G3BP Ab (to label SGs) and DAPI (blue). Data information: Scale bars, 10 µm.


E . Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations , calculated as the ratio between TRAPPC2 ( mean intensity ) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL . Mean ± s . e . m . , n = 40 - 60 cells per experiment , N = 3 . ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations, calculated as the ratio between TRAPPC2 (mean intensity) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL. Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


F . Representative images of Sec24C localization at SGs ( stained for G3BP ) in TRAPPC3 - KD and TRAPPC2 - KD cells treated with SA . Depletion of the entire TRAPP complex ( via TRAPPC3 depletion ) or of only TRAPPC2 reduces Sec24C recruitment . Graph , quantification of Sec24C at SGs , calculated as in ( E ) . Mean ± s . e . m . , n = 40 - 60 cells per experiment , N = 3 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . Data information : Scale bars , 10 µm .

O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC3-KD and TRAPPC2-KD cells treated with SA. Depletion of the entire TRAPP complex (via TRAPPC3 depletion) or of only TRAPPC2 reduces Sec24C recruitment. Graph, quantification of Sec24C at SGs, calculated as in (E). Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ****p< 0.0001, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.


G . Representative images of Sec24C localization at SGs ( stained for G3BP ) in TRAPPC2 - KO cells treated with SA . Data information : Scale bars , 10 µm .

O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O

G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA. Data information: Scale bars, 10 µm.


A . TRAPP and COPII associate more stably with SGs than with ERES . The membrane association of Sec24C was evaluated in non - permeabilized or permeabilized cells with or without SA treatment , as indicated . G3BP was used as an SG marker . Left panel insets , G3BP staining in non - permeabilized cells . Dashed white lines show the outline of permeabilized SA - untreated cells . Blue , DAPI . Data information ; Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A. TRAPP and COPII associate more stably with SGs than with ERES. The membrane association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI. Data information; Scale bars, 10 µm.


B . Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs . HeLa cells overexpressing GFP - Sar1H79G were treated with SA and immunostained for Sec24C , TRAPPC2 , and eIF3 , as indicated . Blue , DAPI . Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP - Sar1H79G - expressing cells . Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES , expressed as a percentage of the non - transfected ( NT ) cells . Mean ± s . e . m . n = 60 - 70 , three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


C . Effect of Sec31A depletion on TRAPPC2 recruitment to SGs . Cells were mock - treated or KD for Sec31 , treated with SA , and then immunostained for TRAPPC2 and eIF3 as indicated . Graphs , quantification of TRAPPC2 at ERES and SGs after SA treatment . Mean ± s . e . m . , one representative experiment ; n = 60 - 80 . * * p < 0 . 001 , * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


D . Effect of Sec31A depletion on Sec24C recruitment to SGs . Cells were mock - treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 ( to stain ERES , top panels ) and for eIF3 ( bottom panels ) . Insets show eIF3 . Graphs , quantification of Sec24C at ERES and in SGs after SA treatment . n = 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


A . Cells were treated with 273 kinase inhibitors ( 10 µM ) from the Sellekchem library and with SA , and Sec24C localization in G3BP puncta ( % of total G3BP area ) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control ( cells treated with SA alone ) . Gray dashed line , mean of control ; gray box , control standard deviation ( ± 18 . 2 % ) ; red line , threshold of positive hits ; red box : positive hits . B . Enrichment Analysis of the positive hits ; for each class of inhibitor , the enrichment ( in percentage ) of compounds falling into positive hits and the enrichment ( in percentage ) in the total number of compounds was calculated and expressed as a ratio .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio.


C . Evaluation of CDK kinase activity upon SA treatment . Western blot analysis of phosphoSer - CDK substrates in control , SA ( 300µM , 30 min ) , Dinaciclib ( 10 µM , 180 min ) and SA + Dinaciclib ( 150 min Dinaciclib and 30 min SA ) . CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition . Left , Western blot , right : Ponceau was used as loading control . Image , one representative experiment out of 3 independent replicates .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.


D . Drug specificity from high ( + + + + ) to low ( + ) of the different CDKs as described by Selleckchem , and Sec24C recruitment to SGs as a percentage of control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control.


E . Analysis of Sec24C recruitment to SGs in control and CDK1 + 2 - KD HeLa cells . Different levels of CDK1 + 2 KD were achieved by transfecting cells with different amounts of siRNA for different times ( 2 nM 24 h ; 5 nM 24 h ; 5 nM 48 h ; 20 nM 48 h ) to give 50 , 30 , 20 and 10 % , respectively , of the CDK1 + 2 levels in the control . CDK1 + 2 levels measured by qRT - PCR and Sec24C recruitment to SGs were evaluated from parallel cultures . Sec24C recruitment to SGs was performed by automated microscopy as described in ( A ) and is expressed as a percentage of mock ( CTRL , set at 100 % ) ± s . d . N = 8 .

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Analysis of Sec24C recruitment to SGs in control and CDK1+2-KD HeLa cells. Different levels of CDK1+2 KD were achieved by transfecting cells with different amounts of siRNA for different times (2 nM 24 h; 5 nM 24 h; 5 nM 48 h; 20 nM 48 h) to give 50, 30, 20 and 10%, respectively, of the CDK1+2 levels in the control. CDK1+2 levels measured by qRT-PCR and Sec24C recruitment to SGs were evaluated from parallel cultures. Sec24C recruitment to SGs was performed by automated microscopy as described in (A) and is expressed as a percentage of mock (CTRL, set at 100%) ± s.d. N=8.


F . Representative images of HeLa cells treated with the three best hits ( Flavopiridol , SNS - 032 , Dinaciclib ; 1µM 150 min ) and then exposed to SA ( 300 µM , 30 min ) , followed by immunostaining for Sec24C and G3BP . Scale bar , 10 µm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O

F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm.


G . HeLa cells were treated with Flavopiridol , SNS - 032 , or Dinaciclib for 150 min at the indicated concentrations , subsequently treated with SA ( 300 µM , 30 min ) , and then immunostained for Sec24C and G3BP and imaged by OPERA . BS - 181 , a specific CDK7 inhibitor , was used as negative control . Sec24C localization to SGs is expressed as a percentage of the control ( cells treated with SA alone ) . Dashed red line , mean of control ; yellow box , standard deviation of control ( ± 9 . 8 % ) , N = 12 .

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12.


H . Cells were treated as in ( G ) and immunostained for TRAPPC2 and eIF3 . Scale bar , 10 µm . The graph shows the quantification of TRAPPC2 localization in SG puncta ( mean intensity ) . Data are mean ± s . e . m . expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol , SNS - 032 or Dinaciclib treatment compared to the control ( cells treated with SA alone ) . N = 3 , three independent experiments , n = 60 - 80 cells per experiment . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


A . Immunofluorescence images of Sec24C and TRAPPC2 in vehicle - treated and SNS - 032 - treated ( 1 µM , 3 h ) cells . Graphs show the quantification of Sec24C and TRAPPC2 at ERES ( mean intensity ) normalized in SNS - 032 - treated cells relative to vehicle - treated cells ( set as 100 % ) . Mean ± s . e . m . of three independent experiments . * * p < 0 . 05 , * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information : Scale bars , 10 µm .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed t-test. Data information: Scale bars, 10 µm.


B . HeLa cells were treated with Dinaciclib ( 1 µM , 3 h ) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency . Upper panels , non - permeabilized ( NP ) control cells ; middle panels , permeabilized control cells ; lower panels , permeabilized Dinaciclib - treated cells . Data information : Scale bars , 10 µm .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

B. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Data information: Scale bars, 10 µm.


C . Quantification of Sec24C membrane association after CDK inhibitor treatment . Digitonin - permeabilized cells treated with vehicle ( CTRL ) or the indicated CDK inhibitor ( 1 µM , 3 h ) were immunostained for Sec24C . The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug - treated cells is expressed as fold change compared to the control ; n = 60 - 80 ; mean ± s . e . m . of three independent experiments * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


D . HeLa cells transfected with HA - hnRNPK were left untreated ( Steady state ) or treated with SA ( 300µM , 30 min ) or SA + Dinaciclib ( 10 µM , 150 min ) and then immunostained with anti - HA and anti - G3BP antibodies . Data information Scale bars , 10 µm .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies. Data information Scale bars, 10 µm.


E . TRAPPC2 interacts with hnRNPK . ( Top ) SDS - PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti - TRAPPC2 Ab or IgG , and ( bottom ) after immunoprecipitation with an anti - HA Ab from cells transfected ( + ) or not ( - ) with hnRNPK - HA detected with the indicated antibodies .

O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

E. TRAPPC2 interacts with hnRNPK. (Top) SDS-PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti-TRAPPC2 Ab or IgG, and (bottom) after immunoprecipitation with an anti-HA Ab from cells transfected (+) or not (-) with hnRNPK-HA detected with the indicated antibodies.


F . Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3 . Data information : Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O

F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3. Data information: Scale bars, 10 µm.


G . Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP . Data information : Scale bars , 10 µm .

G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP. Data information: Scale bars, 10 µm.


H . Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated ( CTRL ) and hnRNPK KD cells . n > 100 , N = 3 mean ± s . d . * * * p < 0 . 001 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated (CTRL) and hnRNPK KD cells. n>100, N=3 mean ± s.d. ***p<0.001.****p<0.0001, Student's unpaired two-tailed t-test.


A . HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA ( 30 min , 300 µM ) , and immunostained for Sec24C or TRAPPC2 and eIF3 . Scale bar 10 µm .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm.


B . Analysis of the proliferation status of the cells after 8 h starvation in HBSS . Top , representative images of starved and non - starved ( CTRL ) cells using EdU incorporation ( see Materials and Methods ) . Scale bar 100 µm . Bottom , quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells , N = 3 , mean ± s . d .

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

B. Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Top, representative images of starved and non-starved (CTRL) cells using EdU incorporation (see Materials and Methods). Scale bar 100 µm. Bottom, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells, N=3, mean ± s.d.


C . HeLa cells were seeded at different confluency , treated with SA and stained for Sec24C and G3BP as an SG marker . Scale bar , 10 µm . Flow cytometry ( FACS ) analysis ( right panels ) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency . The graph shows quantification of Sec24C mean intensity in SG puncta ( normalized for the cytosolic Sec24C ) at the indicated cell confluency . Mean ± s . e . m . of a representative experiment out of 5 biological replicates . n = 50 - 80 . ns : not significant ; * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


D . Differentiation of podocytes . Western blot of growing and differentiated podocytes . Synaptopodin ( SYNPO ) was used as a differentiation marker , GAPDH as a loading control .

O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

D. Differentiation of podocytes. Western blot of growing and differentiated podocytes. Synaptopodin (SYNPO) was used as a differentiation marker, GAPDH as a loading control.


E . Growing and differentiated podocytes were treated with SA ( 300 µM , 30 min ) and stained for TRAPPC2 or Sec24C and eIF3 . Scale bar , 10 µm . The graphs show quantification of TRAPPC2 and Sec24C ( mean intensity ) in SGs . Mean ± s . e . m . of three independents experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed t-test.