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 .

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(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 ) .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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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 .

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(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 .

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(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 % .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ] ) .

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(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 ] ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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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.


( A ) Transwell was performed to evaluate invasion abilities of HCT116 after transfection of IDH1 K224R or knockout of IDH1 . was performed by counting stained that invaded to lower chamber under a microscopy . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For A statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

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(A) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after transfection of IDH1 K224R or knockout of IDH1. Quantification was performed by counting the stained cells that invaded to the lower chamber under a light microscopy. 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 a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( B ) Three - dimension imaging was to assessed velocity of movement . 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 one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

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(B) Three-dimension imaging analysis was to assessed velocity of cell movement. 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 one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( C ) migration ability was evaluated by wound healing in HCT116 . was performed by measuring smallest clearance of wound . 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 a two - way ANOVA followed by Tukey ' s post hoc . * P < 0 . 05 * * P < 0 . 01 .

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(C) Cell migration ability was evaluated by wound healing assay in HCT116 cells. Quantification was performed by measuring the smallest clearance distance of the wound. 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 a two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.


( D ) Colony formation ability after deacetylation of IDH1 K224 or knockout of IDH1 . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

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(D) Colony formation ability after deacetylation of IDH1 K224 or knockout of IDH1. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( E ) cycle distribution was assessed flow cytometrically after treatment with IDH1 K224R or knockout of IDH1 . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For E , statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc . * P < 0 . 05 * * P < 0 . 01 .

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(E) Cell cycle distribution was assessed flow cytometrically after treatment with IDH1 K224R or knockout of IDH1. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For E, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( A ) Pictures of nude mice their tumors after inoculation was shown in two subgroups . ( B ) volumes of each group during growth process . ( C ) measured from xenograft model . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . Statistical significance was assessed with a two - unpaired Student ' s t - . * * P < 0 . 01 .

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(A) Pictures of the nude mice and their tumors after inoculation was shown in two subgroups. (B) Tumor volumes of each group during the tumor growth process. (C) Tumor weight measured from the xenograft model. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01.


( D ) Pictures of livers in nude mice their respective representative of tissues by hematoxylin - eosin ( HE ) . Data information : results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . Statistical significance was assessed with a two - unpaired Student ' s t - . * * P < 0 . 01 .

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(D) Pictures of livers in nude mice and their respective representative images of the tissues by hematoxylin-eosin (HE) staining. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01.


( A ) Expression of IDH1 K224Ac in human CRC ( n = 90 ) CRN ( n = 90 ) tissue specimens , assessed by immunohistochemistry . CRC , colorectal carcinoma samples ; CRN , matched adjacent non - cancerous colorectal tissue specimens .

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(A) Expression levels of IDH1 K224Ac in human CRC (n=90) and CRN (n=90) tissue specimens, assessed by immunohistochemistry. CRC, colorectal carcinoma samples; CRN, matched adjacent non-cancerous colorectal tissue specimens.


( B ) Kaplan - Meier survival curves of CRC cases with elevated ( n = 49 ) reduced ( n = 41 ) IDH1 K224Ac . CRC , colorectal carcinoma samples . overall survival was analyzed by log - rank using Kaplan - Meier method .

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(B) Kaplan-Meier survival curves of CRC cases with elevated (n=49) and reduced (n=41) IDH1 K224Ac. CRC, colorectal carcinoma samples. The overall survival was analyzed by the log-rank test using the Kaplan-Meier method.


A . Schematic depiction of Caenorhabditis elegans phasmid cilia ( left ) . Two sensory cilia are aligned into phasmid channel ( dashed box enlarged in middle ) . Each cilium contains a ciliary base , a middle segment ( m . s . ) , a distal segment ( d . s . ) . Right shows an inverted Hessian - SIM image of GFP - tagged dynein - 2 heavy chain CHE - 3 in cilia , in which were resolved . Scale bar : 1 μm .

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A. Schematic depiction of the Caenorhabditis elegans phasmid cilia (left). Two sensory cilia are aligned into the phasmid channel (the dashed box and enlarged in the middle). Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). Right shows an inverted fluorescence Hessian-SIM image of GFP-tagged dynein-2 heavy chain CHE-3 in cilia, in which individual puncta were resolved. Scale bar: 1 μm.


B . Frame series of an anterograde moving IFT - particle from a 7xGFP : : CHE - 3 worm . Localization precisions are shown under each frame . Time in millisecond ( ms ) ; Scale bar , 100 nm .

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B. Frame series of an anterograde moving IFT-particle from a 7xGFP::CHE-3 worm. Localization precisions are shown under each frame. Time in millisecond (ms); Scale bar, 100 nm.


C . Localization precisions ( mean ± SD ) of tracks from multi - GFP - labeled worms . number of GFP copies was estimated as following : dynein - 2 heavy chain of CHE - 3 forms a dimer , 14 = 2 x 7 ; IFT - particle subunit CHE - 2 is also a dimer , 14 = 2 x 7 ; IFT - particle subunit DYF - 11 can be a tetramer , 12 = 4 x 3 ; genetic cross combined 7xGFP : : CHE - 3 with DYF - 11 : : 3XGFP , 26 = 2 x 7 + 4 x 3 .

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C. Localization precisions (mean ± SD) of tracks from multi-GFP-labeled worms. The number of GFP copies was estimated as the following: the dynein-2 heavy chain of CHE-3 forms a dimer, 14 = 2 x 7; IFT-particle subunit CHE-2 is also a dimer, 14 = 2 x 7; IFT-particle subunit DYF-11 can be a tetramer, 12 = 4 x 3; the genetic cross combined 7xGFP::CHE-3 with DYF-11::3XGFP, 26 = 2 x 7 + 4 x 3.


D . representative diagram shows displacement of cargo within middle ciliary segment . y - axis grid lines are spaced 16 nm . CHE - 3 tagged with 7x GFP was used for tracking experiments . Trace was analyzed using SIC fitting

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D. The representative diagram shows the displacement of the cargo within the middle ciliary segment. The y-axis grid lines are spaced 16 nm. Protein CHE-3 tagged with 7x GFP was used for tracking experiments. Trace was analyzed using the SIC fitting algorithm


E . Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms . plus minus displacements are defined as upward " jumping " or downward " jumping " markers used for tracking experiments are CHE - 3 tagged with 7x GFP , CHE - 2 tagged with 7x GFP , or CHE - 3 tagged with 7x GFP plus DYF - 11 tagged with 3x GFP .

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E. Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms. The plus and minus displacements are defined as the upward "jumping" or downward "jumping" Protein markers used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-2 tagged with 7x GFP, or CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP.


A . Frame series shows off - axis movement of an IFT - particle ( # 1 ) . A neighboring particle ( # 2 ) serves as a fiduciary marker .

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A. Frame series shows the off-axis movement of an IFT-particle (#1). A neighboring particle (#2) serves as a fiduciary marker.


B . Representative two - dimensional IFT - tracks from WT , kinesin - II ( klp - 11 ) , osm - 3 null . Red violet circles indicate start end of tracks , respectively . used for tracking experiments are CHE - 3 tagged with 7x GFP , or DYF - 11 tagged with 3x GFP .

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B. Representative two-dimensional IFT-tracks from WT, kinesin-II (klp-11), and osm-3 null animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.


C . Time - averaged mean - square displacement ( MSD ) versus lag time ( tlag ) of 10 - 15 tracks from WT IFT - kinesin mutant . used for tracking experiments are CHE - 3 tagged with 7x GFP , CHE - 3 tagged with 7x GFP plus DYF - 11 tagged with 3x GFP , CHE - 2 tagged with 7x GFP , or DYF - 11 tagged with 3x GFP .

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C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and IFT-kinesin mutant animals. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP, CHE-2 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.


E . Average deviation angles in 10 - 15 tracks from WT mutant . Comparisons were performed with WT .

E. Average deviation angles in 10-15 tracks from WT and mutant animals. Comparisons were performed with the WT.


F . Representative tracks of recombinant C . elegans kinesin - II OSM - 3 - kinesin moving along suspended MTs in optical trap .

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F. Representative tracks of the recombinant C. elegans kinesin-II and OSM-3-kinesin moving along suspended MTs in the optical trap assay.


G . Representative of middle ciliary segments from WT IFT - kinesin mutant . Red arrowheads indicate ectopic singlet microtubules . Blue arrowheads indicate ectopic doublet microtubules . schematics below depict phenotype of each image .

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G. Representative TEM images of the middle ciliary segments from WT and IFT-kinesin mutant animals. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image.


B . Representative two - dimensional IFT tracks from WT OSM - 3 ( 1 or 3xGS ) . Red violet circles indicate start end of tracks , respectively .

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B. Representative two-dimensional IFT tracks from WT and OSM-3(1 or 3xGS) animals. Red and violet circles indicate the start and end of the tracks, respectively.


C . Time - averaged mean - square displacement ( MSD ) versus lag time ( tlag ) of 10 - 15 tracks from WT mutant worms .

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C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and mutant worms.


A . Histogram of anterograde IFT speeds of 3xGFP : : CHE - 3 at middle ( top ) distal ( bottom ) ciliary segments ( top ) . Comparisons were performed between WT osm - 3 mutants . * * *

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A. Histogram of anterograde IFT speeds of 3xGFP::CHE-3 at the middle (top) and distal (bottom) ciliary segments (top). Comparisons were performed between the WT and osm-3 mutants. ***


B . Schematic depiction of C . elegans amphid phasmid cilia . Each cilium contains a ciliary base , a middle segment ( m . s . ) , a distal segment ( d . s . ) . C . Amphid ( top ) phasmid ( bottom ) cilia in WT osm - 3 GS knock - in . Cilia are marked with 7xGFP : : CHE - 3 . Arrowheads indicate ciliary base . Arrows indicate junctions between middle distal segments .

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B. Schematic depiction of the C. elegans amphid and phasmid cilia. Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). C. Amphid (top) and phasmid (bottom) cilia in the WT and osm-3 GS knock-in animals. Cilia are marked with 7xGFP::CHE-3. Arrowheads indicate the ciliary base. Arrows indicate the junctions between the middle and distal segments.


D . Cilium ( mean ± SD ) in WT osm - 3 mutant . Comparisons were performed between WT osm - 3 mutants .

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D. Cilium length (mean ± SD) in WT and osm-3 mutant animals. Comparisons were performed between the WT and osm-3 mutants.


E . Quantifications of dye - filling defects from WT osm - 3 mutant .

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E. Quantifications of dye-filling defects from WT and osm-3 mutant animals.


F . Representative of middle ciliary segments from osm - 3 ( 1xGS ) osm - 3 ( 3xGS ) worms . Red arrowheads indicate ectopic singlet microtubules . Blue arrowheads indicate ectopic doublet microtubules . schematics below depict phenotype of each image .

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F. Representative TEM images of the middle ciliary segments from osm-3(1xGS) and osm-3(3xGS) worms. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image.


( A , B ) Knockdown of hnRNPA3 increases poly - GA expression while expressions of EGFP are not altered upon knockdown of hnRNPs . control ( " x0 " ) vector lacks G4C2 repeats but still contains 5 ' flanking region 3x TAG . ANOVA , Dunnett .

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(A, B) Knockdown of hnRNPA3 increases poly-GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control ("x0") vector lacks the G4C2 repeats but still contains the 5' flanking region and 3x TAG. ANOVA, Dunnett.


( C , D ) Overexpression of hnRNPA3 suppresses poly - GA expression . n = 2 experiments performed in duplicates . ANOVA , Tukey .

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(C, D) Overexpression of hnRNPA3 suppresses poly-GA expression. n=2 experiments performed in duplicates. ANOVA, Tukey.


( E ) Increased repeat upon hnRNPA3 knockdown . EGFP are only slightly increased as compared to of repeat . n = 2 experiments performed in duplicates . ANOVA , Dunnett .

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(E) Increased repeat RNA upon hnRNPA3 knockdown. EGFP RNA levels are only slightly increased as compared to levels of the repeat RNA. n=2 experiments performed in duplicates. ANOVA, Dunnett.


( F , G ) Knockdown of hnRNPA3 increases foci . n = 3 replicates ; two paired t - , Scale bar = 20 μm . graphs are shown as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F, G) Knockdown of hnRNPA3 increases RNA foci. n=3 replicates; two tailed paired t-test, Scale bar=20 μm. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.


( A - C ) Rescue of repression of poly - GA repeat by wild type ( wt ) hnRNPA3 hnRNPA2 but not by binding mutant hnRNPA3DxD . ( B ) n = 3 experiments performed in duplicates ; ( C ) n = 2 experiments performed in duplicates . ANOVA , Tukey .

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(A-C) Rescue of repression of poly-GA and repeat RNA by wild type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n=3 experiments performed in duplicates; (C) n= 2 experiments performed in duplicates. ANOVA, Tukey.


( D - F ) Rescue of repression of poly - GA repeat by hnRNPA3WT but not M9 - NLS deletion mutant . ( E ) n = 3 experiments performed in duplicates ; ( F ) n = 4 experiments performed in duplicates . ANOVA , Tukey . Graphs are shown as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( See also Figure EV1 ) .

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(D-F) Rescue of repression of poly-GA and repeat RNA by hnRNPA3WT but not the M9-NLS deletion mutant. (E) n=3 experiments performed in duplicates; (F) n=4 experiments performed in duplicates. ANOVA, Tukey. All Graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (See also Figure EV1).


( A ) poly - GA co - localize with p62 - positive deposits .

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(A) poly-GA aggregates co-localize with p62-positive deposits.


( B ) Redistribution of TDP - 43 to cytoplasm in a poly - GA / poly - GR double - positive . Note that nuclei of poly - GA / poly - GR double positive are frequently disrupted .

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(B) Redistribution of nuclear TDP-43 to the cytoplasm in a poly-GA/poly-GR double-positive cell. Note that nuclei of poly-GA/poly-GR double positive cells are frequently disrupted.


( C ) Knockdown of hnRNPA3 increases repeat expression . n = 2 experiments performed in duplicates . ANOVA , Dunnett .

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(C) Knockdown of hnRNPA3 increases repeat RNA expression. n=2 experiments performed in duplicates. ANOVA, Dunnett.


( D ) Filter trap probed with anti - FLAG , anti - myc or anti - HA antibodies reveal abundant DPR production . ( E ) Relative expression level of poly - GA , poly - GP poly - GR normalized to GR . n = 3 experiments , ANOVA , Tukey .

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(D) Filter trap assay probed with anti-FLAG, anti-myc or anti-HA antibodies reveal abundant DPR production. (E) Relative expression level of poly-GA, poly-GP and poly-GR normalized to GR. n=3 experiments, ANOVA, Tukey.


( F ) Expression of three DPRs is increased upon knockdown of hnRNPA3 . Signals from knockdown with siRNA # 8 were normalized to 1 . n = 3 experiments , ANOVA , Dunnett .

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(F) Expression of all the three DPRs is increased upon knockdown of hnRNPA3. Signals from knockdown with siRNA#8 were normalized to 1. n=3 experiments, ANOVA, Dunnett.


( G ) Co - expression of two / three different DPRs in single is increased upon hnRNPA3 knockdown . n = 3 replicates , ANOVA , Dunnett . graphs are shown as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bars = 20μm . ( see also Figure EV2 EV3 ) .

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(G) Co-expression of two/three different DPRs in single cells is increased upon hnRNPA3 knockdown. n=3 replicates, ANOVA, Dunnett. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Scale bars=20μm. (see also Figure EV2 and EV3).


Rathippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA ( shA3 ) or a control shRNA ( shCtrl ) tag RFP . Three days after transduction ( DIV3 + 3 ) , neurons were transfected with ( G4C2 ) 80HIGH ( + 0 ) analyzed at ( DIV3 + 7 ) . ( A ) Neurons were fixed , immunostained imaged by confocal microscopy . Double immunofluorescence for poly - GA ( green ) tag RFP ( red ) . Nuclei were labeled with DAPI . Scale bar represent 20 μm .

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Rathippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (A) Neurons were fixed, immunostained and imaged by confocal microscopy. Double immunofluorescence for poly-GA aggregates (green) and tag RFP (red). Nuclei were labeled with DAPI. Scale bar represent 20 μm.


Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA ( shA3 ) or a control shRNA ( shCtrl ) tag RFP . Three days after transduction ( DIV3 + 3 ) , neurons were transfected with ( G4C2 ) 80HIGH ( + 0 ) analyzed at ( DIV3 + 7 ) . ( B ) Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA .

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Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (B) Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA.


Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA ( shA3 ) or a control shRNA ( shCtrl ) tag RFP . Three days after transduction ( DIV3 + 3 ) , neurons were transfected with ( G4C2 ) 80HIGH ( + 0 ) analyzed at ( DIV3 + 7 ) . ( C ) Poly - GA were detected in a filter trap using an anti - Flag antibody . ( D ) amounts of poly - GA were quantified are presented as fold change of signals from neurons treated with control shRNA or repeat construct . Means ± SD of three independent experiments are shown . * p < 0 . 05 by a Student ' s t - . n = 3 replicates .

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Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (C) Poly-GA aggregates were detected in a filter trap assay using an anti-Flag antibody. (D) The amounts of poly-GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. * p<0.05 by a Student's t-test. n= 3 replicates.


( B ) Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers . Actin was used as a loading control .

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(B) Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers. Actin was used as a loading control.


( C ) Knockdown of hnRNPA3 increases foci in derived from C9orf72 carriers . No G4C2 repeat foci are detected in fibroblast without C9orf72 repeat expansions ( WT ) . Nucleoli were stained with anti nucleolin antibodies ( green ) nuclei were stained with DAPI ( blue ) . ( D ) of relative frequency of foci positive ( foci positivity ) ( fold change ) . n = 3 experiments for each case . ( E ) of number of foci per foci - positive ( fold change ) . n = 3 experiments for each case . ( D , E ) average foci number of non - treated ( N / T ) fibroblast was normalized to 1 in each case . Bars indicate mean . points indicate mean values obtained from a single experiment ( 49 - 214 were counted in single experiment ) . Error bars indicate 95 % CI , ANOVA , Tukey - Kramer HSD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers. No G4C2 repeat RNA foci are detected in fibroblast without C9orf72 repeat expansions (WT). Nucleoli were stained with anti nucleolin antibodies (green) and nuclei were stained with DAPI (blue). (D) Quantification of the relative frequency of RNA foci positive cell (RNA foci positivity) (fold change). n=3 experiments for each case. (E) Quantification of the number of RNA foci per RNA foci-positive cell (fold change). n=3 experiments for each case. (D, E) The average foci number of non-treated (N/T) fibroblast was normalized to 1 in each case. Bars indicate mean. Individual points indicate mean values obtained from a single experiment (49-214 cells were counted in single experiment). Error bars indicate 95%CI, ANOVA, Tukey-Kramer HSD, *p<0.05, **p<0.01, ***p<0.001.


( F ) Representative presentation of increased foci in C9orf72 carriers upon lentiviral - mediated hnRNPA3 knockdown . ( G ) of number of foci ( fold change ) . 3 cases were analyzed . Single points indicate an average obtained from 29 - 30 per case . Color code labels values obtained in fibroblasts derived from three . Two - t - . Mean ± . Scale bar = 10 μm .

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(F) Representative presentation of increased RNA foci in C9orf72 carriers upon lentiviral-mediated hnRNPA3 knockdown. (G) Quantification of the number of RNA foci (fold change). 3 cases were analyzed. Single points indicate an average obtained from 29-30 cells per case. Color code labels values obtained in fibroblasts derived from the three individual patients. Two-tailed t-test. Mean ± SEM. Scale bar= 10 μm.


( A ) Double immunofluorescence with anti - GA ( green ) anti - hnRNPA3 ( red ) antibodies of granular layer of dentate gyrus of a control case three C9orf72 mutation cases . In C9 mutation cases with low hnRNPA3 expression ( # 8 # 1 ) more poly - GA were observed as in cases with high hnRNPA3 ( # 7 ) . Inserts show examples of co - localization of poly - GA hnRNPA3 .

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(A) Double immunofluorescence staining with anti-GA (green) and anti-hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 #1) more poly-GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co-localization of poly-GA and hnRNPA3 aggregates.


( B ) Granular layer neurons of a C9orf72 mutation case with ( arrows ) or without ( orange arrows ) clearance of hnRNPA3 . ( C ) poly - GA are more frequent in C9orf72 mutation cases with lower hnRNPA3 than in those cases with higher hnRNPA3 ( divided by median of hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups ) . Bar graph indicates mean value . Error bars indicate 95 % CI . Single points indicate mean from three per case . Note that difference in GA positivity between both groups remains significant ( p = 0 . 0086 ) after removal of highest outlier in low A3 group . Two - t - . Scale bar = 10 μm .

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(B) Granular layer neurons of a C9orf72 mutation case with (white arrows) or without (orange arrows) nuclear clearance of hnRNPA3. (C) poly-GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean value. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (p = 0.0086) after removal of the highest outlier in the low nuclear A3 group. Two-tailed t-test. Scale bar = 10 μm.


( B ) by micro - CT of mean volumes in KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , 20 post - infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 18 mice , 21 mice , 21 mice , 20 mice , respectively ) .

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(B) Quantification by micro-CT of mean tumor volumes in KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively).


( C ) by micro - CT of total volume per mouse in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 8 , 12 , 16 , 20 post - infection .

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(C) Quantification by micro-CT of total tumor volume per mouse in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection.


( D ) Depictions quantitation of total burden ( total / total lung ) in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 21 after infection . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(D) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 21 weeks after infection. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( E ) Distribution of histological grades in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) at 21 after infection . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(E) Distribution of histological tumor grades in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each) at 21 weeks after infection. For bar charts, data presented as means with error bars representing standard deviations (SDs).


( F ) Assessment of mitotic index of by phosphorylated - histone H3 ( pHH3 ) - positive nuclei density in KP mice LUAD tumors at 21 after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(F) Assessment of the mitotic index of tumor cells by phosphorylated-histone H3 (pHH3)-positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( G ) Nf1 mRNA expression in LUAD sections 21 after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(G) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( H ) Representative hematoxylin eosin ( H & E ) p - Fak1 immunohistochemical ( ) of LUAD sections 21 after infection with control sgTom ( grade 1 depicted ) , sgNf1 . 1 ( grade 3 depicted ) , sgNf1 . 2 ( grade 3 depicted ) , or sgNf1 . 3 ( grade 3 depicted ) ( n = 50 tumors each ) . H & E scale bars ( low - magnification top row = 100 μm , high - magnification bottom row = 250 μm ) ; p - Fak1 scale bars ( low - magnification top row = 250 μm , high - magnification bottom row = 500 μm ) .

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(H) Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm).


( I ) of p - Fak1 signals in LUAD sections 21 after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(I) Quantification of p-Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( J ) of p - Fak1 signals in sgNf1 . 3 LUAD sections analyzed by grade . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(J) Quantification of p-Fak1 IHC signals in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( K ) of Psat1 mRNA expression in LUAD sections 21 after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(K) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( L ) of Psat1 mRNA expression in sgNf1 . 3 LUAD sections analyzed by grade . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(L) Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Western blotting of p53 expression in various p53 / TP53 - WT lines incubated with - intercalating agent doxorubicin ( DOXO , 0 . 2 µg / ml for 6 hours ) to induce p53 stabilization ( n = 3 biological replicates ) .

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(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates).


( B , C ) Western blotting confirming NF1 upregulation p - FAK1 downregulation in ( B ) DOXO - treated PDKN1 ( C ) DOXO - treated PDKN2 transduced with PGK - NF1 ( n = 3 biological replicates ) .

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(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates).


( D ) Western blotting confirming NF1 downregulation p - FAK1 upregulation in DOXO - treated SW1573 transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates).


( E , F ) Western blotting confirming Nf1 downregulation p - Fak1 upregulation in ( E ) DOXO - treated LKR10 ( F ) DOXO - treated LKR13 clones transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).


( G ) FAK1 target gene expression in DOXO - treated PDKN1 transduced with PGK - control or PGK - NF1 ( n = 3 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H , I ) Subcutaneous volumes of DOXO - treated PDKN1 PDKN2 transduced with PGK - control or PGK - NF1 ( n = 18 each ) .

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(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).


( J - L ) Subcutaneous volumes of ( J ) SW1573 , ( K ) LKR10 , ( L ) LKR13 transfected with sgTom or sgNf1 . 3 ( n = 18 each ) .

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(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).


( M ) Depictions quantitation of KrasG12D / + ; p53 + / + ( K - only ) autochthonous burden ( total / total lung ) in pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( N ) of grades in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( O ) of Ki - 67 - positive nuclei per mm2 in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Glucose consumption lactate excretion in KP KPΔNF1 clones normalized by ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( B ) Glutamine consumption in KP KPΔNF1 clones normalized by ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( C ) Relative viability of KP KPΔNF1 after 72 h of GPNA treatment assayed by - titer glo ( relative luminescent units ) ( n = 4 biological replicates ) .

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(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).


( D ) Cumulative population doublings of KP KPΔNF1 cultured with 2 . 0 or 0 . 5 mM glutamine ( n = 4 biological replicates ) .

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(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).


( E ) Two - derived KRAS ; NF1 - mutant LUAD lines ( PDKN1 , PDKN2 ) , as well as control - derived KRAS - mutant / NF1 - WT LUAD ( PDK ) , were passaged for 14 population doublings prior to collection . displays most significantly upregulated genes in PDKN1 PDKN2 lines ( relative to PDK control ) , with degree of upregulation depicted by color as indicated in legend .

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(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.


of expression of six key altered genes in cultured PDKN1 ( passaged for 14 population doublings ) vs . their respective original samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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Quantification of the expression of the six key altered genes in cultured PDKN1 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).


of expression of six key altered genes in cultured PDKN2 ( passaged for 14 population doublings ) vs . their respective original samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

Quantification of the expression of the six key altered genes in cultured PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H ) Western blotting of Psat1 expression in KP KPΔNF1 infected with sgTom or sgPsat1 following selection . GAPDH was used as a loading control .

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(H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control.


( I ) Cumulative population doublings of KP KPΔNF1 after transduction with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


( J - L ) Cumulative population doublings of - derived KRAS - mutant LUAD lines that are either ( J , K ) NF1 - mutant ( PDKN1 PDKN2 ) or ( L ) NF1 - WT ( PDK ) after selection with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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(J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


Relative viability of KP KPΔNF1 clones after CB - 839 treatment ( IC50 : 18 nM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . data points are relative to vehicle - treated controls ( n = 4 biological replicates ) .

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Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).


Relative viability of KP KPΔNF1 clones after AOA treatment ( IC50 : 6 µM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . data points are relative to vehicle - treated controls ( n = 4 biological replicates ) .

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Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).


( D ) Cumulative population doublings of KP KPΔNF1 incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA after 6 d in culture ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( E ) Relative viability of indicated human KRAS - mutant lung cancer lines by trypan blue exclusion . were incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA for 72 h ( n = 4 biological replicates ) . data points are normalized to vehicle - treated lines . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).