G , Representative of Syt1Ab uptake ( magenta ) driven by endogenous network activity in hippocampal neurons ( 18 DIV ) from WT BsnGT mice . VGLUT1 ( green ) marks excitatory presynapses ( upper panels ) VGAT inhibitory ones ( lower panels ) . H . of normalized IF of Syt1Ab uptake in excitatory ( red boxes ) inhibitory ( pink boxes ) synapses on experiments in G . I . of fraction of active ( Syt1Ab - labelled ) excitatory inhibitory synapses on experiments illustrated in G . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . dashed lines in H depict IF in WT that were used for normalization . sample n ( in parentheses ) corresponds to number of analysed ( G , Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - ; * * * p < 0 . 001 . Scale bar is 5 µm in

O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD B-ORGANISM O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and BsnGT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels). H. Quantification of normalized IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in G. I. Quantification of fraction of active (Syt1Ab-labelled) excitatory and inhibitory synapses on experiments illustrated in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in H depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images (G, Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in G


J . Representative of Syt1Ab uptake ( magenta ) upon depolarization with 50 mM KCl . Identical cultures were applied as in G . K . of normalized IF of Syt1Ab uptake on experiments from J . L . of fraction of active excitatory inhibitory synapses in J . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . dashed lines in K depict IF in WT that were used for normalization . sample n ( in parentheses ) corresponds to number of analysed J ) . Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - ; * * * p < 0 . 001 . Scale bar is 5 µm in J .

O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in G. K. Quantification of normalized IF of Syt1Ab uptake on experiments from J. L. Quantification of fraction of active excitatory and inhibitory synapses in J. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in K depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images J). Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in J.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted for WT BsnGT neurons treated with a CDK5 inhibitor roscovitine ( rosc , 100µM , gray pink trace ) or vehicle ( black red trace ) for 30 minutes before stimulation with 40 900 APs in presence of bafilomycin A . C , D . Plots show mean values of RRP ( C ) TRP ( D ) fraction for both genotypes before after treatment . E . Roscovitine treatment has a significantly higher effect on RRP TRP in BsnGT neurons compared to WT . Data information : n corresponding to number of imaging experiments done on 4 independent preparations is given in brackets for each . In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . Significance was assessed by two - way ANOVA with Tukey ´ s multiple comparison ( C , D ) by Student ' s t - ( E ) * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 2µm .

O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT. Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.


A . Representative immunoblot with antibody against CDK5 - dependent pSer551 of Syn1 ( pSer551Syn1 ) total Syn1 total stain used as loading control for of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT BsnGT mice . B , C . of pSer551Syn1 / Syn1 ( in B ) total Syn1 ( in C ) abundance on blots from A . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample n ( in brackets ) corresponds to number of independently processed in B C For statistics , student ' s t - in B C were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice. B, C. Quantification of pSer551Syn1 / Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from A. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C For statistics, student's t-test in B and C were used, *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative of 19 DIV cultured hippocampal neurons from WT BsnGT immunostained for pSer551Syn1 total Syn1 . F . of IF of pSer551Syn1 in synapses from WT , BsnGT , WT treated with roscovitine . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample n ( in brackets ) corresponds to number of independent ( E , quantified from two ( F ) independent cultures per condition . For statistics , one - way ANOVA with Sidak ´ s multiple comparisons in F were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1. F. Quantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT, and WT cells treated with roscovitine. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images (E, quantified from two (F) independent cultures per condition. For statistics, one-way ANOVA with Sidak´s multiple comparisons test in F were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in


G . Representative of 21 DIV hippocampal WT BsnGT neurons at baseline activity , upon network activity silencing ( TTX , 1 µM , 72 h ) or in conditions of increased activity ( BIC , 30 µM , 48 h ) H . of experiment in G . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample n ( in brackets ) corresponds to number of independent , G ) quantified from four ( H ) independent cultures per condition . For statistics two - way ANOVA with Tukey ´ s multiple comparisons in H were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm in G .

O O O O O O B-TISSUE O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h) H. Quantification of experiment in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images , G) quantified from four (H) independent cultures per condition. For statistics two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in G.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted from WT BsnGT hippocampal neurons treated with calcineurin inhibitor FK506 . Data information : Scale bar is 2 µm .

O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506. Data information: Scale bar is 2 µm.


C , D . of RRP TRP fractions in WT BsnGT neurons from experiment in A , B . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets for each reflect number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison for C D significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A, B. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test for C and D significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative Western blot on P2 fractions prepared from hippocampal tissue of WT BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 total SNAP25 . F . of blot in E shows lower pThr138SNAP25 to total SNAP25 IF in BsnGT compared to WT . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets for each reflect number of per genotype used for WB in F Statistical significance was assessed using Student ' s t - in F , significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O B-GENEPROD B-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25. F. Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student's t-test in F, significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted for WT BsnGT neurons without treatment ( black red ) upon treatment with adenylyl cyclase activator forskolin ( gray pink ) or PKA inhibitor H89 ( grey orange ) . Data information : scale bar is 2 µm in

O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange). Data information: The scale bar is 2 µm in A


C , D . of RRP TRP fraction from experiment in A B . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample is given in brackets corresponds to number of analysed independent imaging experiments performed on three independently prepared culture batches in ( C D ) , statistical significance was assessed in C , D using two - way ANOVA with Tukey ´ s multiple comparison * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C, D. Quantification of the RRP and TRP fraction from experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), The statistical significance was assessed in C, D using two-way ANOVA with Tukey´s multiple comparison test *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative immunoblot with antibody against pSer9Syn1 total Syn1 total stain of hippocampal P2 fraction from WT BsnGT mice prepared in presence of KN 93 to isolate PKA - dependent regulation of pSer9Syn1 . F , G . of pSer9Syn1 / Syn1 ( in F ) Syn1 ( in G ) on blots from E . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample is given in brackets corresponds to number of analysed independent imaging experiments samples prepared from in F G , statistical significance was assessed in F G using Student ' s t - as is depicted in graphs as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1. F, G. Quantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from E. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G, The statistical significance was assessed in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001.


H . Representative of hippocampal neurons from WT BsnGT mice labelled with antibodies against pSer9Syn1 total Syn1 . Neurons were pre - treated ( 4 µM , 1hr ) with KN93 to isolate PKA - dependent phosphorylation then treated in addition with forskolin or H89 before fixation . I . of in H . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample is given in brackets corresponds to independent visual fields obtained from 2 independent culture preparations in I . statistical significance was assessed in I using two - way ANOVA with Tukey ´ s multiple comparison as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . scale bar is 5 µm in H .

O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation. I. Quantification of staining in H. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in I using two-way ANOVA with Tukey´s multiple comparison test as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 5 µm in H.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted for untreated WT BsnGT neurons ( black red ) cultures treated with of 6 - Bnz - cAMPS ( 50 µM , 1 hour ; gray pink ) rolipram ( 1 µM , 30 min , grey orange ) . Data information : Scale bar is 2µm .

O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and BsnGT neurons (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram (1 µM, 30 min, light grey and orange). Data information: Scale bar is 2µm.


C , D . Quantifications of RRP TRP fractions in experiment in A B . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets corresponds to number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison in C , D : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C, D. Quantifications of RRP and TRP fractions in experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test in C, D : *p≤0.05, **p < 0.01, ***p< 0.001.


H . of pSer145PDE4B to total PDE4B from experiment in I . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets corresponds to used for sample preparation Statistical significance was assessed using Student ' s t - in H is given as : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student's t-test in H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001.


I . Representative immunoblot with antibody against CDK5 - dependent pSer145PDE4B total PDE4B on hippocampal tissue from WT BsnGT .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O B-TISSUE O O O B-GENEPROD O

I. Representative immunoblot with antibody against CDK5-dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals.


Representative of normal injured acinar . Scale bar , 2μm . Right ( insets ) - higher magnification of boxed regions from left show emergence of double - membraned autophagic vesicles engulfing mature granules , mitochondria , rough ER 24h after cerulein ( CER ) injury . Scale bar , 500nm . Arrowheads mark degradation events without double membranes . Graph of number of acinar granules per in vehicle vs . 24h CER treatment . Each data point = total granules in a in a single counted from one mouse . Red indicates mean . Graph of number of degradation events scored in vehicle vs . 24h CER treatment . Each data point = total events in a in a single counted from one mouse . Red indicates mean .

O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

Representative TEM images of normal and injured acinar cells. Scale bar, 2μm. Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after cerulein (CER) injury. Scale bar, 500nm. Arrowheads mark degradation events without double membranes. Graph of the number of acinar cell granules per cell in vehicle vs. 24h CER treatment. Each data point=total granules in a cell in a single cell counted from one mouse. Red line indicates mean. Graph of the number of degradation events scored in vehicle vs. 24h CER treatment. Each data point=total events in a cell in a single cell counted from one mouse. Red line indicates mean.


mRNA expression by qRT - PCR of Rab5 , Rab7a , Rab7b , Rab9 . Significance determined by one - way ANOVA , Tukey post hoc . Each data point represents mean of triplicate technical replicates from a single mouse , n = 3 - 4 mice in independent experiments . Error bars denote standard deviation .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O

mRNA expression by qRT-PCR of Rab5, Rab7a, Rab7b, and Rab9. Significance determined by one-way ANOVA, Tukey post hoc. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Error bars denote standard deviation.


Top - Representative RAB7 expression in base of stomach units ( ZCs ) in vehicle or 24h HDT by immunohistochemistry . Arrowheads point to RAB7 vesicle . Scale bar 20µm . Bottom - Representative ATF3 expression in base of stomach units ( ZCs ) by immunohistochemistry . ATF3 appears in nuclei following 24h high - dose tamoxifen ( HDT ) . Scale bar 20µm . Stomach unit base outlined by dashed black .

O O O B-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O O O O

Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line.


Western blot of ATF3 expression over HDT CER timecourses with β - Actin loading control .

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Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control.


Top - Representative RAB7 in pancreatic acinar . Arrowheads indicate RAB7 vesicles . Scale bar 20µm . Bottom - Representative ATF3 expression in pancreatic acinar . Nuclei express ATF3 after 24h cerulein ( CER ) injury . Scale bar 20µm .

O O O O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

Top - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm.


ChIP - PCR on mouse stomach tissue at vehicle 6 hour HDT . probed with ATF3 , Histone H3 antibodies , or a Rabbit IgG control . Also included is 2 % input water to control for contamination . Rab7b amplicon is from a conserved , putative ATF3 - binding site in first intron . Amplicon from Atf3 is a positive control from a previously characterized ATF3 - binding site ; negative control is from a site in Rab9a lacking putative ATF3 binding motifs .

B-EXP_ASSAY O B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O

ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.


mRNA expression of ATF3 by qRT - PCR in control , ATF3 knockdown ( KD ) , ATF3 overexpression ( OE ) AGS lines generated using a PiggybacTM transposon system . Significance by one - way ANOVA , Dunnett post - hoc . Datapoint = mean of technical triplicates in a single mouse . n = 3 mice . Error bars denote standard deviation . mRNA expression of RAB7B by qRT - PCR in AGS lines from 3A . Statistics plotting as for panel A .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

mRNA expression of ATF3 by qRT-PCR in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cell lines generated using a PiggybacTM transposon system. Significance by one-way ANOVA, Dunnett post-hoc. Datapoint = mean of technical triplicates in a single mouse. n=3 mice. Error bars denote standard deviation. mRNA expression of RAB7B by qRT-PCR in AGS cell lines from 3A. Statistics and plotting as for panel A.


Top - Immunofluorescence for RAB7 ( red ) DAPI ( blue , nuclei ) in control , ATF3 knockdown ( KD ) , ATF3 overexpression ( OE ) AGS . Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue . Bottom - Immunofluorescence for ATF3 ( purple ) Cathepsin D ( CTSD , green , lysosomes ) in AGS . Scale bar , 20µm .

O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O

Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm.


Representative immunofluorescence of gastric chief after vehicle or 24h HDT treatment stained for Pepsinogen C ( PGC , red , granules ) MAP1LC3B ( green , autophagy marker ) . Top row is wild - type , bottom row is Atf3 − / − . Scale bar , 50μm . Stomach unit base outlined by dashed .

O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O

Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line.


Transmission electron micrograph of zymogenic chief ( ZCs ) after 24h vehicle or HDT treatment in WT ( top ) Atf3 − / − mice ( bottom ) . Scale bar , 1μm . Arrowheads indicate components being degraded . of degradation events per counted from EM of ZCs after 24h HDT treatment in WT Atf3 − / − mice as shown in panel 3D . Each data point = total granules in a in a single section with from 2 separate mice plotted together ( data point colors indicate from a single mouse ) . Red indicates mean across mice .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O

Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.


Representative immunofluorescence of gastric units after vehicle or 72h high - dose tamoxifen ( HDT ) treatment stained for GIF ( red , granules ) , GSII ( green , neck SPEM marker ) , BRDU ( , proliferation ) DAPI ( blue , nuclei ) . Top row is wild - type , bottom row is Atf3 − / − . Yellow arrowheads point to areas of base dropout where ZCs have been lost . Scale bar , 50μm . Stomach units outlined by dashed .

O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_TYPE B-DISEASE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O

Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.


Representative immunohistochemistry of - Caspase 3 in pancreatic acinar marking apoptosis in vehicle or 24h cerulein ( CER ) treatment . Top row is WT , bottom row is Atf3 − / − . Scale bar , 50μm . Apoptosis quantified following 24h CER injury by number of - Caspase 3 + from a 40x field . Each data point is mean of positive in 4 - 5 40x fields in an mouse , n = 3 mice per treatment group . Significance by unpaired two - t - . Error bars denote standard deviation .

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-TISSUE B-CELL_TYPE O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.


Representative immunohistochemistry of BrdU in pancreatic acinar marking proliferation in vehicle or 7 days of repeated cerulein injury . Top row is WT , bottom row is Atf3 − / − . Scale bar , 50μm . of proliferation at peak ADM as counted by number of BrdU + from a 20x field following 7D cerulein treatment . Plotting statistical as per panel F .

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Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.


Serial sections of human tissue . Representative immunohistochemistry of GSII ( top ; spasmolytic polypeptide - expressing metaplasia [ SPEM ] marker neck ) counterstained with hematoxylin ( blue ) eosin ( pink ) . Representative of ATF3 ( bottom ) counterstained with eosin ( pink ) . Tissue was taken from regions adjacent to gastric adenocarcinoma . Scale bars left - 50μm right - 20μm . Arrowheads indicate strongly expressing ATF3 corresponding region in same labeled with GSII in a serial tissue section . GSII at base of gastric in of stomach indicate transition to SPEM .

O O O B-ORGANISM O O O B-EXP_ASSAY O B-GENEPROD O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O B-CELL_TYPE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-TISSUE O O B-TISSUE O O O B-DISEASE O

Serial sections of human tissue. Representative immunohistochemistry of GSII (top; spasmolytic polypeptide-expressing metaplasia [SPEM] marker and neck cells) counterstained with hematoxylin (blue) and eosin (pink). Representative staining of ATF3 (bottom) counterstained with eosin (pink). Tissue was taken from regions adjacent to gastric adenocarcinoma. Scale bars left - 50μm right - 20μm. Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section. GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM.


( A ) abundance is normalized signal ( LFQ ) for a divided by its molecular . Specificity ( enrichment ) is of LFQ in MIA40FLAG fraction to control samples . LFQ for that were not detected in control samples was arbitrarily set to 1 for calculation purposes . LFQ , Label - Free - ; M . W . , molecular .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight.


( B ) Flp - In T - REx 293 with induced MIA40FLAG expression were solubilized , affinity purification of MIA40FLAG was performed . Fractions were analyzed by SDS - PAGE Western blot . Load : 2 . 5 % . Eluate : 100 % . Unbound : 2 . 5 % .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %.


( C ) Flp - In T - REx 293 with induced MIA40FLAG expression were solubilized , affinity purification of MIA40FLAG was performed . Eluate fractions were solubilized under reducing ( DTT ) or non - reducing ( IAA ) conditions analyzed by SDS - PAGE Western blot . DTT , dithiothreitol ; IAA , iodoacetamide . Eluate : 100 % .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.


( D ) extracts from Flp - In T - REx 293 with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification . Load eluate fractions were analyzed by reducing SDS - PAGE Western blot . Load : 2 . 5 % . Eluate : 100 % .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.


( A ) Schematic representation of thiol trapping . Mitochondria were solubilized in sample buffer with either dithiothreitol ( DTT ) , iodoacetamide ( IAA ) , or 4 - acetamido - 4 - maleimidylstilbene - 2 , 2 - disulfonic acid ( AMS ) . samples were analyzed by SDS - PAGE Western blot .

(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.


( B ) Indirect thiol trapping . Mitochondria were pretreated with IAA as indicated to block free cysteine residues , disulfide bonds were subsequently reduced by DTT . Mitochondria were solubilized in sample buffer with AMS .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O

(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.


( C ) Localization of mitochondrial analyzed by limited degradation by proteinase K in intact mitochondria ( 250 mM sucrose ) , mitoplasts ( 5 mM sucrose ) , mitochondrial lysates ( 1 % Triton X - 100 ) . samples were analyzed by SDS - PAGE Western blot . Mitos , mitochondria ; Sup , post - mitochondria supernatant ; OM , outer ; IM , inner ; IMS , intermembrane space .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O

(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.


( D ) N - SIM super - resolution of one Z - stack ( 0 . 15 μm ) orthogonal section ( XYZ ) of HeLa or HeLa that stably expressed COA7 - HA transfected with different markers TOMM20 - DsRed ( OM ) , COX8A - DsRed ( IM ) , matrix targeted photoactivatable GFP ( Matrix target ) labeled with anti - HA , anti - COA7 Smac / Diablo ( IMS ) antibodies . picture represents majority population of from three independent experiments . Scale bar = 2 μm , scale bar in magnified insert = 0 . 5 μm . panel shows Pearson ' s coefficient in a co - localized volume of different subcompartment combinations with anti - HA or anti - COA7 . data are expressed as a mean ± SD ( n = 5 ) . * * * p < 0 . 001 [ IMSxEnd vs . IMxEnd p = 0 . 0001 ; IMSxEnd vs . MxEnd p = 0 . 0002 ] , * * * * p < 0 . 0001 [ OMxHA vs . IMSxHA p < 0 . 0001 ; IMSxHA vs . IMxHA p < 0 . 0001 ] ( one - way ANOVA ) . M , matrix ; End , endogenous COA7 ; HA , COA7 - HA .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

(D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.


( A ) Mitochondria were isolated from that were transfected with a plasmid that encoded COA7HIS or an empty vector . Mitochondria were solubilized analyzed by reducing SDS - PAGE Western blot . Mitos , mitochondria .

O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O

(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria.


( B ) Radiolabeled [ 35S ] TIMM8A [ 35S ] COX19 precursors were imported into mitochondria that were isolated from that were transfected with a plasmid that encoded COA7HIS or an empty vector . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 30 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( C ) Mitochondria were isolated from that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing ( DTT ) non - reducing ( IAA ) conditions analyzed for of MIA40 by Western blot .

O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O

(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.


( D ) from HEK293 transfected with empty plasmid or RESA1HIS were modified with PEG - PCMal . Control were pretreated with thiol oxidizing agent diamide ( DAM ) or reductant DTT . samples were analyzed by SDS - PAGE Western blot .

O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.


( E ) Flp - In T - REx 293 induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector . affinity purification of MIA40FLAG was performed , eluate fractions were analyzed by SDS - PAGE Western blot .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(E) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.


Radiolabeled [ 35S ] COA7 ( A ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 induced to express MIA40FLAG . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


Radiolabeled [ 35S ] TIMM8A ( B ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 induced to express MIA40FLAG . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

Radiolabeled [ 35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( C ) Mitochondria were isolated from Flp - In T - REx 293 induced to express MIA40FLAG control . samples were analyzed by SDS - PAGE Western blot . Mitos , mitochondria .

(C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria.


Radiolabeled [ 35S ] COA7 ( D ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 induced to express ALRFLAG control . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological experiments were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) .

B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

Radiolabeled [35S] COA7 (D) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).


Radiolabeled [ 35S ] TIMM8A ( E ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 induced to express ALRFLAG control . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological experiments were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) .

Radiolabeled [35S] TIMM8A (E) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).


( F ) Mitochondria were isolated from Flp - In T - REx 293 induced to express ALRFLAG control . samples were analyzed by SDS - PAGE Western blot .

(F) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot.


( A ) extracts were isolated from HeLa transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides . samples were analyzed by reducing SDS - PAGE Western blot .

O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(A) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.


( B ) extracts were isolated from HeLa transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides . samples were analyzed by reducing SDS - PAGE Western blot .

(B) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.


( D ) extracts were isolated from HEK293 MIA40 WT / WT WT / Del53 - 60 . samples were analyzed by SDS - PAGE Western blot .

O O O O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(D) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by SDS-PAGE and Western blot.


( E ) extracts were isolated from HEK293 MIA40 WT / WT WT / Del53 - 60 transfected with a plasmid that encoded MIA40 or an empty vector . samples were subjected to reducing SDS - PAGE Western blot .

O O O O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(E) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells transfected with a plasmid that encoded MIA40 or an empty vector. The samples were subjected to reducing SDS-PAGE and Western blot.


Radiolabeled [ 35S ] COA7 ( F ) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT / WT WT / Del53 - 60 . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

Radiolabeled [35S]COA7 (F) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


Radiolabeled [ [ 35S ] TIMM8A ( G ) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT / WT WT / Del53 - 60 . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

Radiolabeled [ [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( B ) extracts were isolated from HEK293 that were transfected with a plasmid that encoded wildtype or mutant COA7 . samples were analyzed by reducing SDS - PAGE Western blot .

O O O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(B) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The samples were analyzed by reducing SDS-PAGE and Western blot.


( C ) fractions were prepared from HEK293 that were transfected with a plasmid that encoded wildtype or mutant COA7 . fractions were analyzed by reducing SDS - PAGE Western blot . T , total ; C , cytosol ; M , mitochondria .

O O O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O

(C) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The fractions were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.


( D ) HEK293 that transiently expressed wildtype or mutant COA7HIS were solubilized , affinity purification of COA7HIS was performed . samples were analyzed by reducing non - reducing SDS - PAGE Western blot . Load : 3 % . Eluate : 100 % .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized, and the affinity purification of COA7HIS was performed. The samples were analyzed by reducing and non-reducing SDS-PAGE and Western blot. Load: 3 %. Eluate: 100 %.


( E ) Equal amounts of wildtype mutant radiolabeled [ 35S ] COA7 precursors were imported into mitochondria isolated from HEK293 . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to wildtype COA7 at 45 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(E) Equal amounts of wildtype and mutant radiolabeled [35S]COA7 precursors were imported into mitochondria isolated from HEK293 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to wildtype COA7 at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( B ) HEK293 that transiently expressed wildtype or mutant COA7HIS were treated with CHX for indicated times , extracts were isolated . samples were analyzed by reducing SDS - PAGE Western blot .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(B) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot.


( D ) HEK293 that transiently expressed wildtype or mutant COA7HIS were treated with CHX / or MG132 for indicated times , extracts were isolated . samples were analyzed by reducing SDS - PAGE Western blot . CHX , cycloheximide .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and/or MG132 for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot. CHX, cycloheximide.


( F ) fractions were prepared from HEK293 that transiently expressed mutant COA7HIS were treated with MG132 . samples were analyzed by reducing SDS - PAGE Western blot .

O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(F) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132. The samples were analyzed by reducing SDS-PAGE and Western blot.


( A ) extracts were isolated from immortalized - derived skin fibroblasts that were treated with indicated concentrations of MG132 , bortezomib , or carfilzomib . samples were analyzed by reducing SDS - PAGE Western blot .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(A) Cellular protein extracts were isolated from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot.


( B ) fractions were prepared from immortalized - derived skin fibroblasts that were treated with indicated concentrations of MG132 , bortezomib , or carfilzomib . samples were analyzed by reducing SDS - PAGE Western blot . T , total ; C , cytosol ; M , mitochondria .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O

(B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.


( A ) IV activity was assessed by oxidation of cytochrome c in digitonized extracts from immortalized - derived skin fibroblasts control healthy fibroblasts results of three biological replicates were quantified , normalized to control fibroblasts . Data are expressed as a mean ± ( n = 3 , * p = 0 . 01 ) .

O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

(A) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient-derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified, and normalized to control fibroblasts. Data are expressed as a mean ± SEM (n=3, *p=0.01).


( B ) IV activity was assessed by oxidation of cytochrome c in digitonized extracts from immortalized - derived skin fibroblasts treated with MG132 ( 1 μM ) , bortezomib ( 20 nM ) , or carfilzomib ( 50 nM ) for 12 h recovered for another 6 h . results of three biological replicates were quantified normalized to DMSO treated samples data are expressed as a mean ± ( n = 3 , * p = 0 . 04 [ DMSO vs MG132 ] , * * p = 0 . 0025 [ DMSO vs Bortezomib ] , * p = 0 . 02 [ DMSO vs Carfilzomib ] ) .

O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O

(B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]).


( C ) Immortalized - derived skin fibroblasts transiently expressing wildtype or mutant COA7 ( COA7 - Y137C COA7 - Ex2 ∆ ) for 48 h were treated with bortezomib ( 20 nM ) during last 12 h . fibroblasts were harvested , IV activity was measured in digitonized extracts . results of three biological replicates were quantified normalized to empty vector - bortezomib treated samples data are expressed as a mean ± ( n = 3 , * p = 0 . 01 [ Empty vector vs COA7 - Wt ] , * p = 0 . 005 [ Empty vector vs COA7 - Y137C ] ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O

(C) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]).


( D ) Mitochondria isolated from control fibroblast were solubilized in digitonin buffer analyzed by 4 - 13 % gel BN - PAGE Western blot . SC , supercomplexes .

O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.


( E ) Immortalized - derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS COA7 - Y137CHIS were solubilized in DDM buffer analyzed by 4 - 13 % gel BN - PAGE Western blot .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(E) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot.


( F ) Mitochondria were isolated from immortalized - derived skin fibroblasts treated with bortezomib ( 10 nM ) for 12 h recovered for another 6 h . Mitochondria were solubilized in digitonin buffer analysed by 4 - 13 % gel BN - PAGE Western blot . SC , supercomplexes .

O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.


( A ) IDH1 acetylation upon treatment with NAM or TSA . Flag - tagged IDH1 was ectopically expressed in HEK293T treated with NAM ( 5 mM ) / or TSA ( 0 . 5 mM ) for indicated time period .

O O O B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(A) IDH1 acetylation levels upon treatment with NAM or TSA. Flag-tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period.


( B ) Five putative lysine residues were mutated . Acetylation of Flag - bead - purified IDH1 were determined by Western blot using a pan - anti - acetyllysine antibody . Relative IDH1 acetylation ratios were calculated after normalizing against Flag .

O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O

(B) Five putative lysine residues were mutated. Acetylation levels of Flag-bead-purified IDH1 were determined by Western blot analysis using a pan-anti-acetyllysine antibody. Relative IDH1 acetylation ratios were calculated after normalizing against Flag.


( C ) Mutation of IDH1 K224R K224Q resulted in altered acetylation using Western Blot .

O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(C) Mutation of IDH1 K224R and K224Q resulted in altered acetylation levels using Western Blot.


( D ) IDH1 catalytic activity of IDH1 K224 mutants in vitro . Wild - type IDH1 K224R , K224Q mutants were expressed in HEK293 . were purified by immunoprecipitation ( IP ) , IDH1 were normalized for , activity assays were performed . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For multiple comparisons in ( D ) , a one - way ANOVA with Newman - Keuls post hoc was used to for statistical significance .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) IDH1 catalytic activity of IDH1 K224 mutants in vitro. Wild-type IDH1 and K224R, K224Q mutants were expressed in HEK293 cells. Proteins were purified by immunoprecipitation (IP), IDH1 levels were normalized for protein, and activity assays were performed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For multiple comparisons in (D), a one-way ANOVA with Newman-Keuls post hoc test was used to test for statistical significance.


( E - F ) Steady - state kinetic of IDH1 variants in HEK293T . Comparison of IDH1 WT K224R K224Q showed that K224 site was important in catalysis . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For ( E - F ) , statistical significance was determined using two - way ANOVA followed by Tukey ' s post hoc . * P < 0 . 05 * * P < 0 . 01 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) Steady-state kinetic analysis of IDH1 and variants in HEK293T cells. Comparison of IDH1 WT and K224R and K224Q showed that the K224 site was important in catalysis. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For (E-F), statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.


( G ) IDH1 directly interacted with SIRT2 but not SIRT1 . SIRT2 deacetylated IDH1 effectively . Flag tagged IDH1 HA tagged SIRT1 or SIRT2 were co - transfected into . acetylation level of Flag - bead - purified IDH1 association between IDH1 SIRT1 or SIRT2 were determined by Western blot .

O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(G) IDH1 directly interacted with SIRT2 but not SIRT1. SIRT2 deacetylated IDH1 effectively. Flag tagged IDH1 and HA tagged SIRT1 or SIRT2 were co-transfected into cells. The acetylation level of Flag-bead-purified IDH1 and the protein association between IDH1 and SIRT1 or SIRT2 were determined by Western blot.


( H ) Co - immunoprecipitation showed endogenous of IDH1 SIRT2 interacted with each other .

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(H) Co-immunoprecipitation showed endogenous proteins of IDH1 and SIRT2 interacted with each other.


( I ) SIRT2 interacted with IDH1 in vitro . GST pull - down with GST - fused SIRT2 Flag - tagged IDH1 .

O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O

(I) SIRT2 interacted with IDH1 in vitro. GST pull-down assay with GST-fused SIRT2 and Flag-tagged IDH1 proteins.


( J ) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O

(J) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot analysis.


( K ) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity . SIRT2 H187Y was a catalytically inactive mutant .

O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O

(K) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity. SIRT2 H187Y was a catalytically inactive mutant.


( A ) Colony formation ability after transfection of SIRT2 plasmids , AGK2 SIRT2 siRNA . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For A statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Colony formation ability after transfection of SIRT2 plasmids, AGK2 and SIRT2 siRNA. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( B ) Transwell was performed to evaluate invasion abilities of HCT116 after overexpression or downregulation of SIRT2 or treated with AGK2 . Stained in lower chambers were quantified . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2. Stained cells in the lower chambers were quantified. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( C ) migration ability was assessed by wound healing in HCT116 . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For figure C , statistical significance was assessed with two - way ANOVA followed by Tukey ' s post hoc . * P < 0 . 05 * * P < 0 . 01 .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Cell migration ability was assessed by wound healing assay in HCT116 cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure C, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.


( D ) cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids , SIRT2 siRNA or AGK2 . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For D , statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids, SIRT2 siRNA or AGK2. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( E ) Immunohistochemical of SIRT2 in representative colorectal tissue specimens is shown ( magnification × 100 ) . Weak for SIRT2 was observed in CRC tissues metastatic tissues , whereas strong was observed in noncancerous tissues .

O O O B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O B-DISEASE O B-DISEASE O O O O O O O O O O

(E) Immunohistochemical analysis of SIRT2 protein in representative colorectal tissue specimens is shown (magnification ×100). Weak cytoplasmic staining for SIRT2 was observed in CRC tissues and metastatic tissues, whereas strong cytoplasmic staining was observed in noncancerous tissues.


( A - B ) IDH1 K224 acetylation level was enhanced with increasing glucose . Flag - IDH1 was overexpressed in treated with increased concentrations of glucose for 6 h . IDH1 were purified by Flag beads , then IDH1 K224 acetylation level was determined by Western blot IDH1 catalytic activity was assessed . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For B statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) IDH1 K224 acetylation level was enhanced with increasing glucose concentration. Flag-IDH1 was overexpressed in cells treated with increased concentrations of glucose for 6 h. IDH1 proteins were purified by Flag beads, and then IDH1 K224 acetylation level was determined by Western blot and IDH1 catalytic activity was assessed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( C - D ) IDH1 K224 acetylation level was upregulated IDH1 activity was weakened with increasing glutamine . Flag - IDH1 was overexpressed in treated with increased glutamine for 6 h . IDH1 were purified by Flag beads , K224 acetylation level of IDH1 was determined by Western blot then IDH1 catalytic activity was evaluated . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For , D statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) IDH1 K224 acetylation level was upregulated and IDH1 activity was weakened with increasing glutamine concentration. Flag-IDH1 was overexpressed in cells treated with increased glutamine for 6 h. IDH1 proteins were purified by Flag beads, and the K224 acetylation level of IDH1 was determined by Western blot analysis and then IDH1 catalytic activity was evaluated. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , D statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. P<0.05 and **P<0.01. n.s. = not significant.


Downregulation of SIRT2 diminished effect of glucose on changing IDH1 K224 acetylation . Flag - tagged IDH1 was overexpressed in HCT116 with or without transient SIRT2 knockdown . were treated with different concentrations of glucose ( E )

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Downregulation of SIRT2 diminished the effect of glucose on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glucose (E)


Downregulation of SIRT2 diminished effect of glutamine on changing IDH1 K224 acetylation . Flag - tagged IDH1 was overexpressed in HCT116 with or without transient SIRT2 knockdown . were treated with different concentrations of glutamine ( F ) .

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Downregulation of SIRT2 diminished the effect of glutamine on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glutamine (F).


( G ) IDH1 K224 deacetylation regulated NADPH / NADP + redox in . In HCT116 stable with IDH1 knockout re - expressing indicated , of NADPH / NADP + in was measured as followed by manufacture ' s instruction . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For , G statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) IDH1 K224 deacetylation regulated cellular NADPH/NADP+ redox in cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of NADPH/NADP+ in cells was measured as followed by manufacture's instruction. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , G statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( H ) IDH1 K224 deacetylation promoted GSH production in HCT116 . In HCT116 stable with IDH1 knockout re - expressing indicated , of GSH / GSSG was assessed as followed by manufacture ' s instruction . 1 Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For H , statistical significance was assessed with one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

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(H) IDH1 K224 deacetylation promoted GSH production in HCT116 cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of GSH/GSSG was assessed as followed by manufacture's instruction. 1 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( I ) IDH1 K224 deacetylation suppressed ROS in HCT116 . ROS was determined in under non - stressed condition or exposed to menadione . * P < 0 . 05 * * P < 0 . 01 Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For figure I , statistical significance was assessed with two - way ANOVA followed by Tukey ' s post hoc . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

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(I) IDH1 K224 deacetylation suppressed cellular ROS levels in HCT116 cells. ROS was determined in cells under non-stressed condition or exposed to menadione. *P<0.05 and **P<0.01 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure I, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( A ) Gene expression profile array in HCT116 . Representative genes with higher fold difference ( up or down ) compared IDH1 K224R with IDH1 WT . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Gene expression profile array analysis in HCT116 cells. Representative genes with higher fold difference (up or down) compared IDH1 K224R cells with IDH1 WT cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition).


( B ) ChIP assays for HIF1α its binding motif . Antibodies anti - IgG anti - HIF1α were used in ChIP assays . qRT - PCR was performed to quantify binding activity . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For B , statistical significance was assessed with a two - unpaired Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) ChIP assays for HIF1α and its binding motif. Antibodies anti-IgG and anti- HIF1α were used in the ChIP assays. qRT-PCR was performed to quantify the binding activity. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B , statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant.


( C - D ) transcriptional activity of SRC promoter was greatly increased after co - expression of HIF1α SRC reporter in wild type . SRC promoter constructs containing a potential HIF1α binding motif ( - 1636 ~ - 1629 bp - 1466 ~ - 1459 bp ) . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For D , statistical significance was assessed with a two - unpaired Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) The transcriptional activity of the SRC promoter was greatly increased after co-expression of HIF1α and SRC reporter in wild type. SRC promoter constructs containing a potential HIF1α binding motif (-1636~-1629 bp and -1466~-1459 bp). Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant.


( E ) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy - HIF1α . In HCT116 stable with IDH1 knockout re - expressing indicated , HIF1α hydroxy - HIF1α was measured by Western blot .

O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(E) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy-HIF1α. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, HIF1α and hydroxy-HIF1α was measured by Western blot assay.


( F ) HCT116 of IDH1 K224Q were cotreated with Octyl - α - KG NAC , expression of HIF1α SRC was further evaluated .

O O O B-CELL_LINE O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O

(F) HCT116 cells of IDH1 K224Q were cotreated with Octyl-α-KG and NAC, the expression of HIF1α and SRC was further evaluated.


HCT116 expression IDH1 K224Q with or without treatment with Octyl - α - KG / or NAC were analyzed for glucose . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For G statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

B-CELL_LINE O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For G statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


HCT116 expression IDH1 K224Q with or without treatment with Octyl - α - KG / or NAC were analyzed for lactate . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For H , statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 . n . s . = not significant .

B-CELL_LINE O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for lactate levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.