Figure 1 ( A ′ ) HeLa cells transfected with pEGFP - RAB11 were fixed in basal conditions and stained for mATG9 . Pearson ' s coefficient : 0 . 452 ± 0 . 034 ( SEM ) . ( A ″ ) HeLa cells were transfected with ATG9L1 - pEGFP , fixed in basal condition , and labeled for RAB11 . Pearson ' s coefficient : 0 . 413 ± 0 . 020 ( SEM ) . ( B ) HeLa cells were incubated for 1 hr at 37 ° C or 18 ° C to reach the correct temperature , loaded with transferrin Alexa - 555 for 1 hr , fixed , and labeled with anti - ATG9 and anti - TGN46 ( marker for Trans Golgi Network ) . Histogram shows Pearson ' s coefficient between ATG9 and transferrin ( Tf ) or TGN46 . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification . ( C ) HeLa cells were incubated in serum - free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa - 555 . Cells were then fixed and labeled for mATG9 and TGN46 . Pearson ' s coefficient between mATG9 and transferrin or TGN46 was quantified . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . Inserts show the merge at higher magnification . ( D ) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy . Pictures are at the same magnification and brightness . Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane . ( E ) HeLa cells were incubated for 1 hr at 4 ° C with ice - cold NHS - LC - Biotin ( Pierce ) solution , rinsed , and lysed in RIPA buffer . Biotinylated proteins were precipitated using streptavidin - agarose beads ( Pierce ) and blotted for mATG9 to assess protein localization at the plasma membrane . ( F ) HeLa cells treated as in ( C ) were processed for immunogold labeling on cryosections and stained with anti - mATG9 antibody . Arrows show clathrin - coated structures . Scale bar in a and c , 150 nm ; scale bar in b , 100 nm . ( G ) HeLa cells transfected with GFP - ATG16L1 were fixed , labeled for mATG9 , and then analyzed by TIRF microscopy . Control cells were cotransfected with GFP - ATG16L1 and Straw - ATG16L1 . Pictures are at the same magnification and brightness . See Figure S1J for quantification . ( H ) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 ( 10 nm ) and mATG9 ( 15 nm ) . Scale bar , 100 nm . ( I ) HeLa cells treated as in ( H ) were processed for triple labeling on cryosections ( clathrin , 5 nm ; ATG16L1 , 10 nm ; and mATG9 , 15 nm ) . Scale bar , 150 nm . ( J ) Table shows quantification of numbers of clathrin - coated structures carrying mATG9 and ATG16L1 alone or together or neither protein . A minimum of 50 clathrin - coated structures were examined from three different experiments . We constructed a 2 × 2 table with 0 or 1 - 3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts . X2 = 10 . 4 , df = 1 , and p = 0 . 001 .

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

Figure 1(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson's coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson's coefficient: 0.413 ± 0.020 (SEM).(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson's coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1-3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.


Figure 2 ( A ) HeLa cells transfected with GFP - ATG16L1 for 24 hr were incubated for 15 min with 2 . 5 μg / ml with HRP - cholera toxin subunit B at 4 ° C and then for 10 min at 37 ° C . Cells were then fixed and treated for immunogold labeling on cryosections . ATG16L1 was detected with anti - ATG16L1 antibody ( 15 nm ) , and cholera toxin was detected using anti - HRP antibody . Scale bar , 150 nm . ( B ) HeLa cells were transiently transfected with GFP - ATG16L1 for 20 hr . Cells in basal conditions were fixed and labeled with anti - RAB11 antibody . ( C ) HeLa cells transfected with mStrawberry - ATG16L1 ( 20 hr ) and starved ( 4 hr ) were processed for immunogold labeling on cryosections . Double labeling was performed with rabbit anti - ATG16L1 ( 10 nm gold ) and rabbit anti - RAB11 ( 15 nm gold ) . Scale bar , 150 nm . Schematic diagram shows membrane outlines from EM . Green lines indicate structures positive for both ATG16L1 and RAB11 , whereas red lines are only positive for RAB11 . Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11 . 281 tubulovesiclular structures from different experiments were analyzed . Error bars , SD . ( D ) HeLa cells were transfected with EGFP - ATG16L1 and mCherry - RAB11 , and 5 min movies were recorded . Representative images from movies are shown . ( E ) HeLa cells were loaded with mouse anti - transferrin receptor antibody ( 5E9C11 ) for 30 min and fixed for immunogold labeling for cryosections . The sample was labeled with rabbit anti - mouse antibody to recognize transferrin receptor ( 5 nm ) , anti - ATG16L1 ( 10 nm ) , and anti - mATG9 ( 15 nm ) . Scale bar , 150 nm . ( F ) HeLa cells were transfected with pmStrawberry - ATG16L1 and ATG9L1 - pEGFP , and 5 min movies were recorded . Representative images from the movies are shown . ( G ) HeLa cells were transfected with pmStrawberry - ATG16L1 and pEGFP - LC3 , starved for 2 hr , fixed , and labeled for mATG9 .

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

Figure 2(A) HeLa cells transfected with GFP-ATG16L1 for 24 hr were incubated for 15 min with 2.5 μg/ml with HRP-cholera toxin subunit B at 4°C and then for 10 min at 37°C. Cells were then fixed and treated for immunogold labeling on cryosections. ATG16L1 was detected with anti-ATG16L1 antibody (15 nm), and cholera toxin was detected using anti-HRP antibody. Scale bar, 150 nm.(B) HeLa cells were transiently transfected with GFP-ATG16L1 for 20 hr. Cells in basal conditions were fixed and labeled with anti-RAB11 antibody.(C) HeLa cells transfected with mStrawberry-ATG16L1 (20 hr) and starved (4 hr) were processed for immunogold labeling on cryosections. Double labeling was performed with rabbit anti-ATG16L1 (10 nm gold) and rabbit anti-RAB11 (15 nm gold). Scale bar, 150 nm. Schematic diagram shows membrane outlines from EM. Green lines indicate structures positive for both ATG16L1 and RAB11, whereas red lines are only positive for RAB11. Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11. 281 tubulovesiclular structures from different experiments were analyzed. Error bars, SD.(D) HeLa cells were transfected with EGFP-ATG16L1 and mCherry-RAB11, and 5 min movies were recorded. Representative images from movies are shown.(E) HeLa cells were loaded with mouse anti-transferrin receptor antibody (5E9C11) for 30 min and fixed for immunogold labeling for cryosections. The sample was labeled with rabbit anti-mouse antibody to recognize transferrin receptor (5 nm), anti-ATG16L1 (10 nm), and anti-mATG9 (15 nm). Scale bar, 150 nm.(F) HeLa cells were transfected with pmStrawberry-ATG16L1 and ATG9L1-pEGFP, and 5 min movies were recorded. Representative images from the movies are shown.(G) HeLa cells were transfected with pmStrawberry-ATG16L1 and pEGFP-LC3, starved for 2 hr, fixed, and labeled for mATG9.


Figure 3 ( A and B ) ( A ) HeLa cells were incubated for 4 hr at 37 ° C or 18 ° C and processed for LC3II western blot . Some cells were incubated overnight with 20 mM NH4Cl in full medium ( to block lysosomal degradation ) at 37 ° C or 18 ° C ( B ) . ( C ) HeLa cells were transfected with pEGFP - LC3 for 20 hr and incubated for 4 hr at 37 ° C or 18 ° C . ( D and E ) ( D ) HeLa cells were transfected with pEGFP - ATG16L1 for 20 hr and incubated for 4 hr at 37 ° C or 18 ° C . The size and the number ( E ) of vesicles were scored ( a minimum of 20 cells were examined for each condition ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ p < 0 . 01 . ( D and E ) ( D ) HeLa cells were transfected with pEGFP - ATG16L1 for 20 hr and incubated for 4 hr at 37 ° C or 18 ° C . The size and the number ( E ) of vesicles were scored ( a minimum of 20 cells were examined for each condition ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ p < 0 . 01 . ( F and G ) ( F ) HeLa cells were transfected with pEGFP - ATG16L1 for 20 hr , incubated for 4 hr at 37 ° C or 18 ° C , and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes . Histogram in ( G ) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment ( Manders ' coefficient ) . Error bar , SEM . NS , not significant ; ∗ ∗ p < 0 . 01 . ( H ) HeLa cells were transfected with pmStrawberry - ATG16L1 for 20 hr , fixed , and labeled for mATG9 . The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37 ° C or 18 ° C . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ p < 0 . 01 . ( I ) HeLa cells were transfected with pEGFP - RAB11 or pEGFP - Myosin Vb tail ( MVb ) and incubated during the last 16 hr with Bafilomycin A1 ( Baf ) or DMSO and processed for LC3 - II western blot . Control cells were transfected with pEGFP empty vector . ( J ) HeLa cells were transfected with ATG16L1 - mStrawberry and RAB11 - EGFP or MVb tail - EGFP . The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions . The inserts show just the ATG16L1 vesicles as quantified in histogram . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( K ) HeLa cells were transfected as in ( C ) and loaded with transferrin Alexa - 647 for 30 min . Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( L ) HeLa cells were transfected as in ( C ) and labeled for mATG9 . Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( M ) HeLa cells were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium , loaded with transferrin Alexa - 488 ( Tf ) . Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( N ) HeLa cells were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium and then fixed and labeled with anti - mATG9 antibody . Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

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

Figure 3(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).(C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C.(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders' coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01.(H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.(J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ∗∗∗p<0.001.(K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.(L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.(M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.(N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


Figure 4 ( A ) HeLa cells were transfected with mStrawberry - ATG16 for 20 hr and treated for 10 min with N - ethylmaleimide ( NEM ; 100 μM ) in full medium , fixed , and stained for mATG9 . Histogram shows correlation between mATG9 and ATG16L1 ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( C and D ) ( C ) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles . The quantification in ( D ) was performed on ten fields ( similar to that shown in the figure ) per experiment . The quantification is expressed as Pearson ' s coefficient . Error bar , SEM . ∗ ∗ p < 0 . 001 .

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

Figure 4(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.(C and D) (C) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles. The quantification in (D) was performed on ten fields (similar to that shown in the figure) per experiment. The quantification is expressed as Pearson's coefficient. Error bar, SEM. ∗∗p < 0.001.


Figure 5 ( A ) HeLa cells were transfected with siRNAs for different R - SNAREs , and we assessed the levels of the autophagy substrate p62 versus actin . ( B ) HeLa cells transfected with control or VAMP3 siRNA ( oligo A ) for 4 days were treated during the last 16 hr with Bafilomycin A1 ( Baf A1 ) or DMSO ( basal ) and processed for LC3 - II western blots . ( C ) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days . The histogram shows the number of vesicles ( LC3 positive ) in a minimum of 30 cells . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( D ) HeLa cells were transfected with VAMP3 - HA for 20 hr and stained for EEA1 or RAB11 . Histogram shows colocalization between VAMP3 - HA and EEA1 or VAMP3 - HA and RAB11 ( Manders ' coefficient ) . Error bar , SEM . ( E ) HeLa cells were transfected for 20 hr with VAMP3 - HA , incubated for 1 hr at 37 ° C or 18 ° C , fixed , and labeled for HA and mATG9 . Histogram shows the percentage of colocalization between VAMP3 with mATG9 ( Manders ' coefficient ) . Error bar , SEM . NS , not significant .

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

Figure 5(A) HeLa cells were transfected with siRNAs for different R-SNAREs, and we assessed the levels of the autophagy substrate p62 versus actin.(B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots.(C) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days. The histogram shows the number of vesicles (LC3 positive) in a minimum of 30 cells. Error bar, SEM. ∗∗∗p < 0.001.(D) HeLa cells were transfected with VAMP3-HA for 20 hr and stained for EEA1 or RAB11. Histogram shows colocalization between VAMP3-HA and EEA1 or VAMP3-HA and RAB11 (Manders' coefficient). Error bar, SEM.(E) HeLa cells were transfected for 20 hr with VAMP3-HA, incubated for 1 hr at 37°C or 18°C, fixed, and labeled for HA and mATG9. Histogram shows the percentage of colocalization between VAMP3 with mATG9 (Manders' coefficient). Error bar, SEM. NS, not significant.


Figure 6 ( A - C ) HeLa cells transfected with control , VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1 - pEGFP and labeled with anti - EEA1 ( for early endosomes ) or anti - RAB11 ( for recycling endosomes ) . The histogram in ( C ) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , SEM . ∗ ∗ ∗ p < 0 . 001 . ( D - F ) HeLa cells were treated as in ( A ) - ( C ) and transfected during the last 20 hr with pEGFP - ATG16L1 and labeled with anti - EEA1 ( early endosomes ) or anti - RAB11 ( recycling endosomes ) . Histogram in ( D ) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , SEM . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

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

Figure 6(A-C) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1-pEGFP and labeled with anti-EEA1 (for early endosomes) or anti-RAB11 (for recycling endosomes). The histogram in (C) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001.(D-F) HeLa cells were treated as in (A)-(C) and transfected during the last 20 hr with pEGFP-ATG16L1 and labeled with anti-EEA1 (early endosomes) or anti-RAB11 (recycling endosomes). Histogram in (D) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001; NS, not significant.


Figure 7 ( A ) HeLa cells transfected with control , VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry - ATG16L1 and labeled for mATG9 . The correlation between mATG9 and ATG16L1 was quantified ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . ( B ) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry - ATG16 , and 5 min movies were recorded . Homotypic fusion events between mStrawberry - ATG16 vesicles were assessed in control and VAMP3 knockdown cells . Error bar , SEM . NS , not significant . ( C ) In vitro fusion assay of ATG16L1 and mATG9 vesicles . HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and , on the fourth day , either transfected with mStrawberry - ATG16L1 or ATG9L1 - pEGFP . PNS of cells expressing the two constructs were mixed for 1 hr at 37C ° and observed by confocal microscopy . Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . The total number of structures per field ( ATG16L1 and ATG9L1 ) is not significantly different in the control and KD samples ( control , 37 ± 3 ; VAMP3 KD , 45 ± 3 ; ± SEM ; p = 0 . 106 ) .

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

Figure 7(A) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry-ATG16L1 and labeled for mATG9. The correlation between mATG9 and ATG16L1 was quantified (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.(B) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry-ATG16, and 5 min movies were recorded. Homotypic fusion events between mStrawberry-ATG16 vesicles were assessed in control and VAMP3 knockdown cells. Error bar, SEM. NS, not significant.(C) In vitro fusion assay of ATG16L1 and mATG9 vesicles. HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and, on the fourth day, either transfected with mStrawberry-ATG16L1 or ATG9L1-pEGFP. PNS of cells expressing the two constructs were mixed for 1 hr at 37C° and observed by confocal microscopy. Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. The total number of structures per field (ATG16L1 and ATG9L1) is not significantly different in the control and KD samples (control, 37 ± 3; VAMP3 KD, 45 ± 3; ± SEM; p = 0.106).


f1 ( a ) WT and Pgam5 KO MEFs evaluated by TEM . The black arrows indicate mitochondria ; ( b ) TEM analysis after 12 h CCCP treatment ; black arrowheads indicate DMS . Inset shows a DMS with an encapsulated mitochondrion . The black arrows indicate mitochondria . Scale bar = 0 . 5 micron ; ( c ) WT and KO MEFs were treated with CCCP for 0 , 3 or 6 h and cytosolic fractions were immunoblotted with anti - LC - 3 , anti - p62 or anti - actin as indicated . ( d ) Similar to c . WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti - COXIV , anti - Tomm20 or anti - actin as indicated . For c and d , the numbers below each lane indicate the fractional band density compared with the 0 h time point , which has been set at 1 . 0 ; ( e ) Confocal photomicrographs of mt - Keima - transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h ( six individual cells from each group were scanned and representative cells are shown ) ; Intracellular mt - Keima excited at 450 nm was shown in green colour , while red colour indicated the excitation by 550 nm in the same cell . Scale bar = 20 μm . ( f ) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO ( − ) or CCCP ( + ) for 3 h and analysed by immunoblot as above . ( Note that mouse PINK1 antibody can only detect the full - length PINK1 ) . Arrow indicates the 63 kDa form of PINK1 . ( g ) Hela cells transduced with either nonspecific ( NS ) or PGAM5 shRNA viruses were treated with CCCP for 3 h , and mitochondrial fractions were immunoblotted for the proteins indicated . Full - length ( * 63 kDa ) and cleaved ( # 54 kDa ) PINK1 are indicated . For f and g , the numbers below indicate the full - length PINK1 band density compared with the first lane that has been set at 1 . 0 ( h ) Confocal micrographs of Hela cells as in g were transfected with PARKIN - YFP ( green ) plasmid before CCCP treatment , then ( i ) treated with CCCP for 12 h . Mitochondria were stained with anti - Tomm20 ( red ) 4 ' , 6 - diamidino - 2 - phenylindole stained nucleus was shown in blue . Co - localization was indicated by white colour . Scale bar = 10 μm . ( j ) Quantification of the co - localization percentage of Tomm20 ( red ) with parkin ( green ) per cell by Imaris software ( n = 7 for NS and PGAM5 shRNA group separately . Results are presented as mean and s . d . values and were analysed by the student t - test ) . All the blots and images are representative of three independent experiments . DMSO , dimethylsulphoxide .

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

f1(a) WT and Pgam5 KO MEFs evaluated by TEM. The black arrows indicate mitochondria; (b) TEM analysis after 12 h CCCP treatment; black arrowheads indicate DMS. Inset shows a DMS with an encapsulated mitochondrion. The black arrows indicate mitochondria. Scale bar=0.5 micron;(c) WT and KO MEFs were treated with CCCP for 0, 3 or 6 h and cytosolic fractions were immunoblotted with anti-LC-3, anti-p62 or anti-actin as indicated.(d) Similar to c. WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0;(e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm.(f) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (−) or CCCP (+) for 3 h and analysed by immunoblot as above. (Note that mouse PINK1 antibody can only detect the full-length PINK1). Arrow indicates the 63 kDa form of PINK1.(g) Hela cells transduced with either nonspecific (NS) or PGAM5 shRNA viruses were treated with CCCP for 3 h, and mitochondrial fractions were immunoblotted for the proteins indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are indicated. For f and g, the numbers below indicate the full-length PINK1 band density compared with the first lane that has been set at 1.0(h) Confocal micrographs of Hela cells as in g were transfected with PARKIN-YFP (green) plasmid before CCCP treatment, then (i) treated with CCCP for 12 h. Mitochondria were stained with anti-Tomm20 (red) 4',6-diamidino-2-phenylindole stained nucleus was shown in blue. Co-localization was indicated by white colour. Scale bar=10 μm. (j) Quantification of the co-localization percentage of Tomm20 (red) with parkin (green) per cell by Imaris software (n=7 for NS and PGAM5 shRNA group separately. Results are presented as mean and s.d. values and were analysed by the student t-test). All the blots and images are representative of three independent experiments. DMSO, dimethylsulphoxide.


f2 ( a ) Mitochondria from MEFs from Parl WT ( + / + ) and KO ( − / − ) mice were treated with the indicated doses of proteinase K ( Prot . K ) for 30 min on ice and then immunoblotted for the indicated proteins . Full - length and cleaved PGAM5 bands are indicated with arrows . ( b ) Pgam5 WT and KO MEFs were transduced with either nonspecific ( NS ) or Parl shRNA and either treated ( + ) or not ( − ) with CCCP for 3 h . Mitochondria were then purified and analysed by immunoblot for the indicated proteins . ( c ) WT PINK1 as well as kinase dead ( KD ) or Parkinson ' s disease - associated PINK1 mutants ( C92F , A168P , H271Q , G309D , L347P , G386S , R464H ) were co - transfected into HEK293T cells with the same amount PGAM5 ( lane 1 - 9 ) or GFP ( lane 10 - 18 ) expression plasmids and total cell lysates were immunoblotting for PINK1 , PGAM5 and actin . The ratio of WT full - length ( 63 kDa ) versus cleaved ( 54 kDa ) PINK1 ( * / # ) is shown . ( d ) Anti - myc immunoprecipitates ( IP ) of lysates from 293T cells transfected with combinations of myc - tagged PGAM5 and PINK1 - V5 with or without CCCP treatment and immunoblotting with anti - PINK1 or anti - myc . ( e ) Four constructs expressing either : ( 1 ) no PGAM5 ( empty vector ) or ( 2 ) amino acids 1 - 98 , ( 3 ) 1 - 110 and ( 4 ) 98 - 289 truncated versions of PGAM5 ( left panel ) . Immunoblot of HEK293T cells expressing PINK1 - GFP and myc - tagged PGAM5 truncation mutants as indicated with anti - PINK1 or anti - myc for the input proteins as well as the anti - GFP IP ( right panel ) . ( f ) Co - transfection of GFP , WT PGAM5 , 2RA mutant ( R98A and R104A ) or the 2HA mutant ( H99A and H105A ) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti - myc IP were immunoblotted with antibodies against PINK1 , PGAM5 or actin . WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after ( g ) DMSO or ( h ) CCCP treatment . Cells were permeabilized with digitonin or Triton X - 100 separately , followed by treatment with the indicated amounts of trypsin on ice for 30 min . Cell lysates were immunoblotted for PINK1 , PGAM5 , Tomm20 or HSP60 as indicated . Full - length ( * 63 kDa ) and cleaved ( # 54 kDa ) PINK1 are shown .

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

f2(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows.(b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins.(c) WT PINK1 as well as kinase dead (KD) or Parkinson's disease-associated PINK1 mutants (C92F, A168P, H271Q, G309D, L347P, G386S, R464H) were co-transfected into HEK293T cells with the same amount PGAM5 (lane 1-9) or GFP (lane 10-18) expression plasmids and total cell lysates were immunoblotting for PINK1, PGAM5 and actin. The ratio of WT full-length (63 kDa) versus cleaved (54 kDa) PINK1 (*/#) is shown.(d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc.(e) Four constructs expressing either: (1) no PGAM5 (empty vector) or (2) amino acids 1-98, (3) 1-110 and (4) 98-289 truncated versions of PGAM5 (left panel). Immunoblot of HEK293T cells expressing PINK1-GFP and myc-tagged PGAM5 truncation mutants as indicated with anti-PINK1 or anti-myc for the input proteins as well as the anti-GFP IP (right panel).(f) Co-transfection of GFP, WT PGAM5, 2RA mutant (R98A and R104A) or the 2HA mutant (H99A and H105A) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti-myc IP were immunoblotted with antibodies against PINK1, PGAM5 or actin.WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.


f4 ( a ) Body weight was measured in 5 - month - old and 12 - month - old female Pgam5 WT and KO mice . Behavioral measurements on 12 - month - old female WT and KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( b ) Open - field tests including time spent mobile , time spent immobile , whole area distance and centre area distance ; Behavioral measurements on 12 - month - old female WT and KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( c ) Gait length , gait frequency in steps / ss and step angle ; ( d ) rearing test , ( e ) akinesia test and ( f ) ladder climb test and ( g ) vertical pole test . T - turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole . T - down indicates the time mice spent to descend to the bottom of pole . Graphs show the mean and s . d . values and were analysed by the student ' s t - test , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 , NS , not significant .

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

f4(a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice.Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (b) Open-field tests including time spent mobile, time spent immobile, whole area distance and centre area distance;Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (c) Gait length, gait frequency in steps/ss and step angle; (d) rearing test, (e) akinesia test and (f) ladder climb test and (g) vertical pole test. T-turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole. T-down indicates the time mice spent to descend to the bottom of pole. Graphs show the mean and s.d. values and were analysed by the student's t-test, *P0.05, **P0.01, ***P0.001, NS, not significant.


f5High - performance liquid chromatography - electrochemical detection quantitation of ( a ) striatal DA and its metabolites ( b ) dihydroxyphenylacetic acid ( DOPAC ) ( c ) Homovanillic acid ( HVA ) and ( d ) 3MT levels at three different time points ( 1 , 2 and 12 - month - old ) in Pgam5 WT or KO mice . ( e ) Representative images of TH - immunohistochemistry from WT and KO mice . The VTA region is indicated . Stereological quantitation of ( f ) TH and ( g ) Nissl - positive neurons in the SNpc ( n = 4 mice per group ) . Error bars represent the mean ± s . e . m . , n = 4 mice per group . ( two - way analysis of variance , * * * P0 . 01 , * P0 . 05 , NS , not significant ) . mo , month old .

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

f5High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice.(e) Representative images of TH-immunohistochemistry from WT and KO mice. The VTA region is indicated. Stereological quantitation of (f) TH and (g) Nissl-positive neurons in the SNpc (n=4 mice per group). Error bars represent the mean±s.e.m., n=4 mice per group. (two-way analysis of variance, ***P0.01, *P0.05, NS, not significant). mo, month old.


f6The 18 - month - old Pgam5 WT and KO mice received either water as control ( n = 4 ) , or 5 mg kg − 1 L - DOPA and 12 . 5 mg kg − 1 Benserazide ( n = 5 ) by intraperitoneal injection . Twenty minutes later , mice were subjected to the open - field test and the centre area time in seconds ( s ) as well as the centre area distance in centimeters ( cm ) were measured as described in the Materials and Methods . Graphs show the mean and s . d . values and were analysed by the student t - test , NS , no significance ; * P0 . 05 ; * * P0 . 01 .

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f6The 18-month-old Pgam5 WT and KO mice received either water as control (n=4), or 5 mg kg−1 L-DOPA and 12.5 mg kg−1 Benserazide (n=5) by intraperitoneal injection. Twenty minutes later, mice were subjected to the open-field test and the centre area time in seconds (s) as well as the centre area distance in centimeters (cm) were measured as described in the Materials and Methods. Graphs show the mean and s.d. values and were analysed by the student t-test, NS, no significance; *P0.05; **P0.01.


Figure 1Flag - INVS interacted with HA - Akt ( lanes 1 - 3 ) , but not with HA - PDK1 ( lanes 4 - 6 ) or HA - PrKA ( lanes 7 - 9 ) in HEK293T cells . Expression levels of HA - Akt , PDK1 , PrKA , and Flag - INVS were similar , as determined by immunoblotting ( HA : anti - HA antibody ; F : anti - Flag antibody ; C : control antibody ) . The results presented ( A - E ) are representative of at least two independent experiments . B . Flag - tagged INVS interacted with HA - tagged Akt1 ( lane 1 - 3 ) , Akt2 ( lane 4 - 6 ) , and Akt3 ( lane 7 - 9 ) in co - immunoprecipitationassays . Expression of each Akt isoform and INVS were similar . C . INVS was co - immunoprecipitated with endogenous Akt from HEK293 cells , in which INVS is also endogenously expressed ( lane 2 ) . D . To determine the Akt domain responsible for interacting with INVS , Akt subfragments were generated . The C - terminus of Akt is the primary domain for INVS interaction ( lanes 7 - 9 ) . Similar levels of each Akt fragment were immunoprecipitated using anti - HA antibody . E . Schematic showing the structure and the functional domains of Flag - tagged full - length and fragmented INVS in mammalian expression vectors ( N - Term : 1 - 421 , Intermediate domain : 421 - 678 , and C - Term : 678 - 1065 ) used in the current study . Intermediate ( lanes 6 - 9 ) and C - terminal ( lanes 10 - 12 ) fragments of INVS were important for interaction with Akt .

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Figure 1Flag-INVS interacted with HA-Akt (lanes 1-3), but not with HA-PDK1 (lanes 4-6) or HA-PrKA (lanes 7-9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A-E) are representative of at least two independent experiments.B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar.C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2).D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7-9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody.E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1-421, Intermediate domain: 421-678, and C-Term: 678-1065) used in the current study. Intermediate (lanes 6-9) and C-terminal (lanes 10-12) fragments of INVS were important for interaction with Akt.


Figure 2A . The indicated recombinant INVS proteins ( lanes 1 - 4 ) were used for IVK . Akt phosphorylates WT ( lane5 ) , 1 - 970 ( lane 6 ) , and 1 - 898 ( lane 7 ) INVS , but failed to phosphorylate 1 - 670 ( lane 8 ) INVS . The indicated recombinant INVS proteins ( lanes 1 - 10 ) were used for IVK . Akt phosphorylates WT ( lane 6 ) and 1 - 898 ( lane7 ) INVS , but failed to phosphorylate other subfragments of INVS ( 1 - 822 , 1 - 746 , and 1 - 670 , in lane 8 , 9 , and 10 , respectively ) , as detected using a phospho - Akt substrate antibody . . C . Using the indicated subfragments of INVS for IVK , we confirmed that Akt phosphorylates 675 - 1065 ( lane 4 ) , but not 675 - 822 ( lane 5 ) , or 675 - 746 ( lane 6 ) INVS . E . Purified recombinant WT or 3A INVS were generated ( top panel , lanes 1 - 4 ) . In IVK , Akt phosphorylated the WT fragment ( 824 - 898 , middle panel , lane 3 ) , but failed to phosphorylate 3A INVS ( middle panel , lane 4 ) , as detected by using a phospho - Akt substrate antibody . F . Akt phosphorylated WT ( lane 10 ) , 1 - 898 ( lane 12 ) and 675 - 1065 ( lane 15 ) INVS , but failed to phosphorylate both full - length and 675 - 1065 , ∆ 727 - 896 , 1 - 857 , or 1 - 822 , 3A INVS ( lanes 16 , 11 , 14 , and 15 , respectively ) , all of which lacked the T864 / S865 / T866 target amino acid .

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Figure 2A. The indicated recombinant INVS proteins (lanes 1-4) were used for IVK. Akt phosphorylates WT (lane5), 1-970 (lane 6), and 1-898 (lane 7) INVS, but failed to phosphorylate 1-670 (lane 8) INVS.The indicated recombinant INVS proteins (lanes 1-10) were used for IVK. Akt phosphorylates WT (lane 6) and 1-898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1-822, 1-746, and 1-670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody..C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675-1065 (lane 4), but not 675-822 (lane 5), or 675-746 (lane 6) INVS.E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1-4). In IVK, Akt phosphorylated the WT fragment (824-898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody.F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.


Figure 3A . Confocal microscopy shows that active Akt ( phospho - S473Akt ) is localized in the basal body of primary cilia ( right side panels show higher magnification ) . B . INVS is localized in the proximal region of the INVS compartment in primary cilia ( INVS compartment , right side panels show higher magnification ) . C . INVS co - localizes with γ - tubulin , a centrosome marker . D . Anti - phospho - Akt substrates stain positively at the basal region of the primary cilia ( arrows ) , as determined by anti - acetylated tubulin ( right side panels show higher magnification ) . E . Silver - intensified immunogold electron microscopy shows that both phospho - Akt ( left side panels ) and INVS ( right side panels ) were localized to the ciliary pocket of primary cilia in the presence of PDGF - AA ( white arrow ) .

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Figure 3A. Confocal microscopy shows that active Akt (phospho-S473Akt) is localized in the basal body of primary cilia (right side panels show higher magnification).B. INVS is localized in the proximal region of the INVS compartment in primary cilia (INVS compartment, right side panels show higher magnification).C. INVS co-localizes with γ-tubulin, a centrosome marker.D. Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin (right side panels show higher magnification).E. Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left side panels) and INVS (right side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrow).


Figure 4PDGF - AA stimulation enhances localization of active Akt ( phospho - S473Akt ) at the basal body of primary cilia ( right side panels show higher magnification ) . Quantification of co - localization of phosphorylated Akt ( green ) and INVS ( red ) at the indicated time points after PDGF - AA stimulation revealed a time - dependent increase . Colocalization was measured by counting yellow pixels ( colocalized area ) using Imaris software ( Bitplane AG ) . Results presented are means ± SE ( n = 31 at time 0 , n = 35 at 1 min . , and n = 33 at 3 min , respectively ) . Three independent experiments were analyzed with similar results . Statistical significance was determined by student ' s t test . Co - immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT - Akt ( second panel , lane1 ) or phospho - mimetic Akt ( T308D / S473D , lane2 ) , but fails to interact with phosphorylation - defective forms of Akt ( T308A / S473A , lane 3 ) . PDGF - AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt ( top panel , comparison between lane 1 and lane 2 , untreated and PDGF - AA - stimulated conditions , respectively ) . Both WT and 3A - INVS , which are localized at the INVS compartment of primary cilia under serum - starved condition ( left side panels , lower panels show higher magnification ) , translocate to the basal body of the cilium after PDGF - AA stimulation ( right side panels , lower panels show higher magnification ) . Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP - INVS ( green ) with γ - tubulin ( red ) in hTERT - RPE1 cells ( n = 28 ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test . Fluorescence intensities of INVS ( green ) , acetylated - α - tubulin ( blue ) , and γ - tubulin ( red ) along the line ( a - b ) are plotted underneath .

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Figure 4PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification).Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student's t test.Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively).Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student's t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.


Figure 5A . PDGF - AA stimulation resulted in the phosphorylation of WT INVS ( top panel , lane 2 ) , but not 3A INVS ( top panel , lane 6 ) . PDGF - AA - stimulated INVS phosphorylation is inhibited by Akt inhibitors ( top panel , LY294002 , MK2206 , and GSK690693 , lanes 3 , 4 , and 5 , respectively ) . B . Myr - Akt , a constitutively active form of Akt , was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified . WT , but not 3A INVS , can be phosphorylated in the presence of Myr - Akt . C . 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS ( top panel , compare lanes 1 and 2 , WT and phospho - defective mutant , respectively ) . Expression of Akt and INVS are shown underneath . D . 3A INVS exhibited weaker dimerization as compared to WT INVS ( top panel , compare lanes 1 and 2 , WT and 3A , respectively ) . Expression of Akt and INVS are shown below each . E . Length of primary cilia in confluent hTERT - RPE cells was analyzed after siRNA - mediated knock down of Akt1 / 2 . Ciliary length decreased in cells expressing siRNA . Results presented are means ± SE of the longitudinal length of acetylated - tubulin ( red ) , a marker of primary cilia , in siRNA - transfected ( green ) hTERT - RPE1 cells ( n = 175 for control siRNA and Akt siRNA , and n = 195 for Akt siRNA plus siRNA resistant Akt , respectively ) . Three independent experiments showed similar results . Statistical significance was analyzed by student ' s t test . Note that re - introduction of siRNA - resistant Akt ( Matsuda - Lennikov et al . , 2014 ) in Akt knockdown cells rescued the effect on ciliary length . F . Expression of 3A INVS ( bottom panels ) but not WT INVS ( middle panels ) inhibited the development of primary cilia as compared to control cells ( top panels ) . Quantification of ciliary length . Results are mean ± SE of longitudinal length of acetylated - tubulin ( blue ) , a marker of primary cilia , in EGFP - INVS ( green ) transfected in hTERT - RPE1 cells ( n = 58 for EGFP vector and for EGFP - INVS WT , and n = 52 for EGFP - INVS 3A mutant , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test .

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

Figure 5A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively).B. Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt.C. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath.D. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each.E. Length of primary cilia in confluent hTERT-RPE cells was analyzed after siRNA-mediated knock down of Akt1/2. Ciliary length decreased in cells expressing siRNA. Results presented are means ±SE of the longitudinal length of acetylated-tubulin (red), a marker of primary cilia, in siRNA-transfected (green) hTERT-RPE1 cells (n=175 for control siRNA and Akt siRNA, and n=195 for Akt siRNA plus siRNA resistant Akt, respectively). Three independent experiments showed similar results. Statistical significance was analyzed by student's t test. Note that re-introduction of siRNA-resistant Akt (Matsuda-Lennikov et al., 2014) in Akt knockdown cells rescued the effect on ciliary length.F. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels).Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


Figure 6Schematic describing the measurement of spindle angle ( θ ) ( Kikuchi et al . , 2010 ) . Z - stack images were obtained from 0 . 45 mm - thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface , which is termed the spindle angle , was calculated . Results presented are scatter plots of the angle ( θ ) of the mitotic spindle ( n = 28 for all samples of vector , HA - WT , - 3D , - 3A , - R899X , - Q671X , and - R603X INVS stably transfected MDCK cells ) . Three independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median test . Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids ( + ) . The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham - transduced cells . Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS , resulted in disorientation of the spindle axis during metaphase . Similar expression of all INVS constructs was verified by immunoblotting ( Appendix figure S4A ) . WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay , which could be reverted by C - terminal truncated INVS ( R899X , Q671X , or Q603X ) , but not by a phospho - mimetic mutant or 3A INVS . Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF / LEF signal and Renilla luciferase signal in HA - INVS transfected 293T cells . Dual - Luciferase reporter ® Assay System ( Promega ) was used and experiments were performed in triplicates . Two independent experiments showed similar results . Statistical significance was determined by student ' s t test . The expression of HA - Dvl ( upper panels ) and the indicated forms of Flag - INVS ( lower panels ) are shown by immunoblots ; α - tubulin was used as an internal control .

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

Figure 6Schematic describing the measurement of spindle angle (θ) (Kikuchi et al., 2010). Z-stack images were obtained from 0.45 mm-thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface, which is termed the spindle angle, was calculated. Results presented are scatter plots of the angle (θ) of the mitotic spindle (n=28 for all samples of vector, HA-WT, -3D, -3A, -R899X, -Q671X, and -R603X INVS stably transfected MDCK cells). Three independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids (+). The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham-transduced cells. Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS, resulted in disorientation of the spindle axis during metaphase. Similar expression of all INVS constructs was verified by immunoblotting (Appendix figure S4A).WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay, which could be reverted by C-terminal truncated INVS (R899X, Q671X, or Q603X), but not by a phospho-mimetic mutant or 3A INVS. Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF/LEF signal and Renilla luciferase signal in HA-INVS transfected 293T cells. Dual-Luciferase reporter® Assay System (Promega) was used and experiments were performed in triplicates. Two independent experiments showed similar results. Statistical significance was determined by student's t test.The expression of HA-Dvl (upper panels) and the indicated forms of Flag-INVS (lower panels) are shown by immunoblots; α-tubulin was used as an internal control.


Figure 7WT INVS - transduced MDCK cells in 3D cultures form uniform round tubules . However , expression of 3A or NPHP2 - related truncated INVS mutants , which lack Akt phosphorylation site ( s ) , disrupts this uniformity . Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector - or WT INVS - transduced MDCK cells ( n = 422 for Vector , n = 398 for HA - WT , n = 401 for - 3D , n = 365 for - 3A , n = 344 for - R899X , n = 331 for - Q671X , and n = 325 for - R603X INVS stably transfected MDCK cells , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test . WT transduced MDCK cells form a relatively smaller lumen ; however , expression of 3A INVS or the indicated NPHP2 - related truncation mutants result in relatively larger acini . Both truncation mutants ( Q671X or R603X ) lack the Akt phosphorylation site and exhibit a larger lumen size . Three independent experiments showed similar results . C . Quantification of normal lumen in cells transduced with WT or mutant INVS . Results presented are mean ± SE of percent normal acini in vector - or WT INVS - transduced MDCK cells ( n = 166 for Vector , n = 204 for HA - WT , n = 233 for - 3D , n = 150 for - 3A , n = 164 for - R899X , n = 128 for - Q671X , and n = 137 for - R603X INVS stably transfected MDCK cells , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t test . D . Size of lumen in cells transduced with WT or mutant INVS is shown . Results presented are scatter plots of acini size ( n = 205 for Vector , n = 143 for HA - WT , n = 131 for - 3D , n = 167 for - 3A , n = 144 for - R899X , n = 128 for - Q671X , and n = 144 for - R603X INVS stably transfected MDCK cells , respectively ) . Two independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median test .

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

Figure 7WT INVS- transduced MDCK cells in 3D cultures form uniform round tubules. However, expression of 3A or NPHP2-related truncated INVS mutants, which lack Akt phosphorylation site (s), disrupts this uniformity. Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=422 for Vector, n=398 for HA-WT, n=401 for -3D, n=365 for -3A, n= 344 for -R899X, n=331 for -Q671X, and n=325 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.WT transduced MDCK cells form a relatively smaller lumen; however, expression of 3A INVS or the indicated NPHP2-related truncation mutants result in relatively larger acini. Both truncation mutants (Q671X or R603X) lack the Akt phosphorylation site and exhibit a larger lumen size. Three independent experiments showed similar results.C. Quantification of normal lumen in cells transduced with WT or mutant INVS. Results presented are mean ±SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=166 for Vector, n=204 for HA-WT, n=233 for -3D, n=150 for -3A, n=164 for -R899X, n=128 for -Q671X, and n=137 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.D. Size of lumen in cells transduced with WT or mutant INVS is shown. Results presented are scatter plots of acini size (n=205 for Vector, n=143 for HA-WT, n=131 for -3D, n=167 for -3A, n=144 for -R899X, n=128 for -Q671X, and n=144 for -R603X INVS stably transfected MDCK cells, respectively). Two independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


Figure 1A Immunofluorescence images of MMS22L foci formation at etoposide ( ETP ) - induced DSBs during S , M and G1 phases of the cell cycle in HeLa cells . Scale bar : 5 μm . B Quantification of a representative experiment from ( A ) shows number of MMS22L foci per nucleus during indicated cell cycle phases ( n = 2 ; nnuclei ≥ 60 ) . C Immunofluorescence images of CPT - induced MMS22L , RPA2 , BRCA2 and RAD51 foci in U2OS cells . Scale bar : 5 μm . D Quantification of a representative experiment ( n = 3 ) from ( C ) shows percentage of γH2AX , RPA2 , BRCA2 and RAD51 foci co - localizing with MMS22L foci ( nnuclei = 30 ; nuclei with ≥ 25 MMS22L foci were analyzed ) .

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

Figure 1A Immunofluorescence images of MMS22L foci formation at etoposide (ETP)-induced DSBs during S, M and G1 phases of the cell cycle in HeLa cells. Scale bar: 5 μm.B Quantification of a representative experiment from (A) shows number of MMS22L foci per nucleus during indicated cell cycle phases (n = 2; nnuclei ≥ 60).C Immunofluorescence images of CPT-induced MMS22L, RPA2, BRCA2 and RAD51 foci in U2OS cells. Scale bar: 5 μm.D Quantification of a representative experiment (n = 3) from (C) shows percentage of γH2AX, RPA2, BRCA2 and RAD51 foci co-localizing with MMS22L foci (nnuclei = 30; nuclei with ≥ 25 MMS22L foci were analyzed).


Figure 2A Electrophoretic mobility shift assay monitoring MMS22L - TONSL binding to ssDNA without RPA and with RPA - coated ssDNA . B Quantification of data as in ( A ) shows percent MMS22L - TONSL binding to ssDNA with or without RPA , n = 2 ; error bars , SEM . C Schematic ( left ) and western blots ( right ) of streptavidin and NiNTA pulldowns of his - tagged MMS22L - TONSL , RPA and biotinylated ssDNA . D Yeast two - hybrid analysis shows the effect of deletion of the amino acids 384 - 522 of MMS22L on the interaction with RPA . The interaction between indicated MMS22L variants fused to LexA DNA - binding domain and RPA1 fused to GAL4 activating domain ( GAD ) is assessed by survival on media containing 3 - aminotriazole ( 3 - AT ) in the absence of histidine and by β - galactosidase activity ( X - Gal ) assays . E Images of MMS22L foci formed in U2OS cells stably expressing wild - type ( WT ) or Δ384 - 522 variants of siRNA - resistant HSS - MMS22L , depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours . Scale bar : 5 μm .

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

Figure 2A Electrophoretic mobility shift assay monitoring MMS22L-TONSL binding to ssDNA without RPA and with RPA-coated ssDNA.B Quantification of data as in (A) shows percent MMS22L-TONSL binding to ssDNA with or without RPA, n = 2; error bars, SEM.C Schematic (left) and western blots (right) of streptavidin and NiNTA pulldowns of his-tagged MMS22L-TONSL, RPA and biotinylated ssDNA.D Yeast two-hybrid analysis shows the effect of deletion of the amino acids 384-522 of MMS22L on the interaction with RPA. The interaction between indicated MMS22L variants fused to LexA DNA-binding domain and RPA1 fused to GAL4 activating domain (GAD) is assessed by survival on media containing 3-aminotriazole (3-AT) in the absence of histidine and by β-galactosidase activity (X-Gal) assays.E Images of MMS22L foci formed in U2OS cells stably expressing wild-type (WT) or Δ384-522 variants of siRNA-resistant HSS-MMS22L, depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours. Scale bar: 5 μm.


Figure 3 ( A - E ) iPOND assays in Hela cells . Experiment schematic ( top ) and resulting immunoblots ( bottom ) are shown in ( A , B , D ) and quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . A Abundance of MMS22L - TONSL at active and HU - stalled replication forks , and fully replicated DNA . ( A - E ) iPOND assays in Hela cells . Experiment schematic ( top ) and resulting immunoblots ( bottom ) are shown in ( A , B , D ) and quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . B , C MMS22L dynamics at stalled replication forks . Quantification shows average relative abundance of indicated proteins detected in immunoblots , n = 3 except of time - point 5 h , n = 1 ; error bars , SEM . ( A - E ) iPOND assays in Hela cells . Experiment schematic ( top ) and resulting immunoblots ( bottom ) are shown in ( A , B , D ) and quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . D , E Effect of MMS22L depletion on RAD51 levels at stalled replication forks . Quantification shows average relative RAD51 abundance , n = 2 ; error bars , SEM . ( A - E ) iPOND assays in Hela cells . Experiment schematic ( top ) and resulting immunoblots ( bottom ) are shown in ( A , B , D ) and quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . D , E Effect of MMS22L depletion on RAD51 levels at stalled replication forks . Quantification shows average relative RAD51 abundance , n = 2 ; error bars , SEM .

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

Figure 3(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.A Abundance of MMS22L-TONSL at active and HU-stalled replication forks, and fully replicated DNA.(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.B, C MMS22L dynamics at stalled replication forks. Quantification shows average relative abundance of indicated proteins detected in immunoblots, n = 3 except of time-point 5 h, n = 1; error bars, SEM.(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.


Figure 4A Representative immunofluorescence images of foci induced by short CPT treatment ( 50 nM , 1 h ) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci . Scale bar : 5 μm . B Boxplot quantification of representative experiment in ( A ) ( n = 3 ; nnuclei ≥ 78 ) . Boxes indicate the 25 - 75 percentile and whiskers the 10 - 90 percentile ; horizontal lines mark the medians . Nuclei with ≥ 25 γH2AX foci ( marker of replication stress sites ) were analyzed . Statistical analysis : Mann - Whitney U test ; * * * P ≤ 0 . 0001 . C , D Electron micrographs of a representative reversed fork ( C ) and a fork with extended ssDNA region at the junction ( D ) ( arrow ) . P , parental duplex ; R , regressed arm . E Frequency of reversed replication forks detected by EM . Similar results were obtained in one independent experiment ; n = 2 . F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h . In brackets , the total number of analyzed molecules is given . Statistical analysis according to Mann - Whitney U test ; * * P ≤ 0 . 01 , n = 2 .

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

Figure 4A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25-75 percentile and whiskers the 10-90 percentile; horizontal lines mark
the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann-Whitney U test; *** P ≤ 0.0001.C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann-Whitney U test; ** P ≤ 0.01, n = 2.


Figure 5A Amylose pulldown of MBP ( control ) or MBP - tagged MMS22L - TONSL and RAD51 . Eluate proteins were detected by silver staining . MT : MBP - MMS22L - GST - TONSL . B Quantitative analysis of RAD51 binding to MMS22L - TONSL such as shown in Figure EV6A . Averages shown , n = 2 ; error bars , SEM . C , D Yeast two - hybrid analysis of interactions between LexA DNA binding domain - tagged MMS22L and indicated proteins fused to GAL4 activating domain ( GAD ) assessed by survival on media containing 3 - AT in the absence of histidine and by X - Gal assays . C , D Yeast two - hybrid analysis of interactions between LexA DNA binding domain - tagged MMS22L and indicated proteins fused to GAL4 activating domain ( GAD ) assessed by survival on media containing 3 - AT in the absence of histidine and by X - Gal assays . E , F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA . The proteins in the input and eluates were detected by western blotting . E , F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA . The proteins in the input and eluates were detected by western blotting . G Streptavidinpulldown of HSS - tagged MMS22L variants stably integrated and overexpressed in U2OS cells . Input and co - precipitated proteins were detected by immunoblotting . H Representative immunofluorescence images of CPT - induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA - resistant variants of HSS - MMS22L treated with 50 nM CPT for 1 hour . Scale bar : 5 μm . I - K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h ( I ; n = 3 ) , 50 nM CPT for 18 h ( J ; n = 3 ) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug ( K ; n = 3 ) . Graphs represent averaged median values ; error bars indicate SEM . Statistical analysis according to one - way ANOVA with Bonferroni post - test ; * * * P ≤ 0 . 001 ; * * P ≤ 0 . 01 ; ns , not significant . Representative immunofluorescence images are shown in ( H ) , Figures EV6G and EV6H , respectively .

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

Figure 5A Amylose pulldown of MBP (control) or MBP-tagged MMS22L-TONSL and RAD51. Eluate proteins were detected by silver staining. MT: MBP-MMS22L-GST-TONSL.B Quantitative analysis of RAD51 binding to MMS22L-TONSL such as shown in Figure EV6A. Averages shown, n = 2; error bars, SEM.C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.G Streptavidinpulldown of HSS-tagged MMS22L variants stably integrated and overexpressed in U2OS cells. Input and co-precipitated proteins were detected by immunoblotting.H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm.I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.


Figure 6B - D Strand exchange assay with ssDNA and RAD51 , and with ( C ) or without ( B ) the addition of MMS22L - TONSL ( 75 nM ) . D Quantification shows averages , n = 2 ; error bars , SEM . E Averages of quantified strand exchange assays with ssDNA , RAD51 ( 270 nM ) and varying concentrations of MMS22L - TONSL or MMS22L . n = 2 ; error bars , SEM . F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA , RAD51 and MMS22L - TONSL or MMS22L . Averages shown , n = 2 ; error bars , SEM . H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA ( 1 nM ) . I A representative immunoblot showing effects of MMS22L - TONSL and MMS22L titration into reactions containing RAD51 , ssDNA ( 1 nM ) and dsDNA ( 10 nM ) , as indicated .

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

Figure 6B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM.E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM.F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.


f1a , alpha - KG extends the lifespan of adult worms in the metabolite longevity screen . All metabolites were given at a concentration of 8 mM . c , Dose - response curve of the α - KG effect on longevity . d , e , α - KG extends the lifespan of worms fed bacteria that have been ampicillin arrested , mean lifespan ( days of adulthood ) with vehicle treatment ( mveh ) = 19 . 4 ( n = 80 animals tested ) , mα - KG = 25 . 1 ( n = 91 ) , P 0 . 0001 ( log - rank test ) ( d ) ; or γ - irradiation - killed , mveh = 19 . 0 ( n = 88 ) , mα - KG = 23 . 0 ( n = 46 ) , P 0 . 0001 ( log - rank test ) ( e ) . OP50 , E . coli OP50 strain . f alpha - KG does not further extend the lifespan of ogdh - 1 RNAi worms , mveh = 21 . 2 ( n = 98 ) , mα - KG = 21 . 1 ( n = 100 ) , P = 0 . 65 ( log - rank test ) .

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f1a, alpha-KG extends the lifespan of adult worms in the metabolite longevity screen. All metabolites were given at a concentration of 8 mM.c, Dose-response curve of the α-KG effect on longevity.d, e, α-KG extends the lifespan of worms fed bacteria that have been ampicillin arrested, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 19.4 (n = 80 animals tested), mα-KG = 25.1 (n = 91), P  0.0001 (log-rank test) (d); or γ-irradiation-killed, mveh = 19.0 (n = 88), mα-KG = 23.0 (n = 46), P  0.0001 (log-rank test) (e). OP50, E. coli OP50 strain.f alpha-KG does not further extend the lifespan of ogdh-1 RNAi worms, mveh = 21.2 (n = 98), mα-KG = 21.1 (n = 100), P = 0.65 (log-rank test).


f2a , DARTS identifies ATP5B as an alpha - KG - binding protein . Red arrowhead , protected band . b , DARTS confirms alpha - KG binding specifically to ATP5B . IB , immunoblot . c , Inhibition of ATP synthase by alpha - KG ( released from octyl alpha - KG ; Supplementary Notes ) . This inhibition was reversible ( data not shown ) . d , e , Reduced ATP levels in octyl alpha - KG - treated normal humanfibroblasts ( * * P = 0 . 0016 , * * * * P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( d ) d , e , Reduced ATP levels in octyl alpha - KG - treated worms ( day 2 , P = 0 . 969 ; day 8 , * P = 0 . 012 ; by t - test , two - tailed , two - sample unequal variance ) ( e ) . RLU , relative luminescence units . f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated cells ( * * * P = 0 . 0004 , * * * * P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( f ) f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated worms ( P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( g ) . h , alpha - KG , released from octyl alpha - KG ( 800 µM ) , decreases state 3 , but not state 4o or 3u ( P = 0 . 997 ) , respiration in mitochondria isolated from mouse liver . The respiratory control ratio is decreased in the octyl alpha - KG - ( 3 . 1 ± 0 . 6 ) versus vehicle - treated mitochondria ( 5 . 2 ± 1 . 0 ) ( * P = 0 . 015 ; by t - test , two - tailed , two - sample unequal variance ) . Oligo , oligomycin ; FCCP , carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ; AA , antimycin A . i , Eadie - Hofstee plot of steady - state inhibition kinetics of ATP synthase by alpha - KG ( produced by in situ hydrolysis of octyl alpha - KG ) . [ S ] is the substrate ( ADP ) concentration , and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol ( vehicle control ) or octyl alpha - KG . alpha - KG ( produced from octyl alpha - KG ) decreases the apparent Vmax ( 53 . 9 to 26 . 7 ) and Km ( 25 . 9 to 15 . 4 ) , by nonlinear regression least - squares fit . c - i , Results were replicated in two independent experiments . Mean ± standard deviation ( s . d . ) is plotted in all cases .

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f2a, DARTS identifies ATP5B as an alpha-KG-binding protein. Red arrowhead, protected band.b, DARTS confirms alpha-KG binding specifically to ATP5B. IB, immunoblot.c, Inhibition of ATP synthase by alpha-KG (released from octyl alpha-KG; Supplementary Notes). This inhibition was reversible (data not shown).d, e, Reduced ATP levels in octyl alpha-KG-treated normal humanfibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d)d, e, Reduced ATP levels in octyl alpha-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units.f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f)f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g).h, alpha-KG, released from octyl alpha-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl alpha-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A.i, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by alpha-KG (produced by in situ hydrolysis of octyl alpha-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl alpha-KG. alpha-KG (produced from octyl alpha-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c-i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.