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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 aggregates co - localize with p62 - positive deposits .

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


( 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 .

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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 RNA 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 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 .

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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 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 .

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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 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 ) .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ( 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 .

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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 ) 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 ) .

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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 ) 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 .

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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 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 .

(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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 ) 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 .

(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 ) 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 .

(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 ) 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 .

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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 ) 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 .

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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 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 ) .

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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 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 ) .

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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 analysis confirming NF1 downregulation and p - FAK1 upregulation in DOXO - treated SW1573 cells 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 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 ) .

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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 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 ) .

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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 tumor volumes of DOXO - treated PDKN1 and PDKN2 cells 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 tumor volumes of ( J ) SW1573 , ( K ) LKR10 , and ( L ) LKR13 cells 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 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 .

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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 ) 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 ) .

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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 ) 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 .

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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 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 ) .

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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 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 ) .

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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 and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell - 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 and KPΔNF1 cells 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 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 .

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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.


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 ) .

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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).


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 ) .

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 analysis of Psat1 expression in KP and KPΔNF1 cells 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 and KPΔNF1 cells 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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).


Quantification by micro - CT of mean tumor volumes in ( B ) CB - 839 - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice and 21 mice , respectively ) . Vehicle - treated KP mice are the same control group for ( B )

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Quantification by micro-CT of mean tumor volumes in (B) CB-839-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B)


Quantification by micro - CT of mean tumor volumes in ( C ) AOA - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice and 21 mice , respectively ) . Vehicle - treated KP mice are the same control group for ( C ) .

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Quantification by micro-CT of mean tumor volumes in (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (C).


Quantification by micro - CT of total tumor volume per mouse in ( D ) CB - 839 - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . Vehicle - treated KP mice are the same control group for ( D )

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Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D)


E ) Quantification by micro - CT of total tumor volume per mouse in , ( E ) AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . Vehicle - treated KP mice are the same control group for ( E ) .

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E) Quantification by micro-CT of total tumor volume per mouse in , (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (E).


Relative responses of KP mice tumors treated with ( F ) CB - 839 , or vehicle starting from week 8 post - infection and measured until week 20 . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of KP mice tumors treated with (F) CB-839, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of KP mice tumors treated with ( G ) AOA , or vehicle starting from week 8 post - infection and measured until week 20 . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of KP mice tumors treated with (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.


( H ) Depictions and quantitation of total tumor burden ( total tumor area / total lung area ) in CB - 839 - , AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 20 weeks post - infection . Vehicle - treated KP mice are the same control group in both boxplots . 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 .

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(H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots. 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.


Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , ( B ) CB - 839 , starting from day 12 and measured until day 27 ( n = 6 tumors per cell line per treatment ) Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, (B) CB-839, starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.


) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , C ) AOA starting from day 12 and measured until day 27 ( n = 6 tumors per cell line per treatment ) Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, C) AOA starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , CB - 839 , or AOA ( D ) the final masses of these tumors . or 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 .

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Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, CB-839, or AOA (D) the final masses of these tumors. or 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.


Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle , ( E ) CB - 839 starting from day 12 and measured until day 27 ( n = 24 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (E) CB-839 starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle , ( F ) AOA starting from day 12 and measured until day 27 ( n = 24 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (F) AOA starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle , ( G ) CB - 839 , with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle, (G) CB-839, with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle ( H ) AOA with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle (H) AOA with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( I ) CB - 839 starting from day 12 and measured until day 30 ( n = 6 tumors per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (I) CB-839 starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle ( J ) AOA starting from day 12 and measured until day 30 ( n = 6 tumors per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle (J) AOA starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of orthotopic patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( K ) CB - 839 starting from day 12 and measured until day 27 ( n = 12 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (K) CB-839 starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of orthotopic patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( L ) AOA starting from day 12 and measured until day 27 ( n = 12 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (L) AOA starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


A - D Proportional distribution of chymotrypsin - like activity of the proteasome across nine tissues from female ( n = 9 ) , and male ( n = 10 ) mice . Proportional distribution of trypsin - like activity of the proteasome across nine tissues from female ( n = 9 ) , and male ( n = 10 ) mice . Graphical representation of color code of tissues analyzed .

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A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed.


Chymotrypsin - like activity of the proteasome across nine tissues in individual mice . Each data point ( n = 9 females and n = 10 males ) indicates data from a single mouse performed in triplicate . Each central line and error bar indicate mean + / - SEM . * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

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Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


trypsin - like activity of the proteasome across nine tissues in individual mice . Each data point ( n = 9 females and n = 10 males ) indicates data from a single mouse performed in triplicate . Each central line and error bar indicate mean + / - SEM . * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


A - D Western blot analysis for K48Ub - linked proteins in spinal cord ( A ) and intestine ( C ) lysates from male and female mice . ( B , D ) Quantification of panels A and C , respectively . Values were normalized to actin as a loading control . Each central line and error bar indicate mean + / - SEM of a biological replicates . Data information : * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

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A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates. Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


E ) Curve of correlation between chymotrypsin - like activity and Ub - K48 linked proteins in the intestine , where variability between mice is observed .

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E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.


F ) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core ( CP ) or assembled with regulatory core ( RP ) in male ( n = 5 ) and females ( n = 5 ) mice in intestine .

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F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine.


G ) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core ( CP ) or assembled with regulatory core ( RP ) in male ( n = 5 ) and female ( n = 5 ) spinal cord .

G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord.


H ) Quantification of the ratio of assembled ( RP - CP ) versus unassembled ( CP ) proteasome in the intestine of males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 per group ) . I ) Quantification of the ratio of RP - CP versus CP proteasome in the spinal cord of males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 per group ) . Data information : * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

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H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


J ) Correlation curve between chymotrypsin - like activity and RP - CP / CP ratio in individual female ( purple ) and male ( blue ) intestine . K ) Correlation curve between chymotrypsin - like activity and RP - CP / CP ratio in individual female ( purple ) and male ( blue ) spinal cord .

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J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.


A - D Comparison of chymotrypsin - like activity from female ( n = 9 ) and male ( n = 10 ) mice in young ( 3 - 5 months old ) or older ( 10 - 15 months old , female ( n = 6 ) and male ( n = 7 ) ) groups . Each point indicates a single mouse . A ) Tissues that did not show significant decline , B ) Tissues that show significant decline in females only , C ) Tissues that show significant decline in males only or D ) in both males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 9 young females , n = 6 older females , n = 10 young males , n = 7 older males ) . P values obtained by unpaired two - tailed students t - test comparing young and older samples are indicated .

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A-D Comparison of chymotrypsin-like activity from female (n=9) and male (n=10) mice in young (3-5 months old) or older (10-15 months old, female (n=6) and male (n=7)) groups. Each point indicates a single mouse. A) Tissues that did not show significant decline, B) Tissues that show significant decline in females only, C) Tissues that show significant decline in males only or D) in both males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=9 young females, n=6 older females, n=10 young males, n=7 older males). P values obtained by unpaired two-tailed students t-test comparing young and older samples are indicated.


B ) Western blot analysis of Ub - K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock . C ) Quantification of panel B . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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B) Western blot analysis of Ub-K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock. C) Quantification of panel B. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group). Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


D ) Western blot analysis of Ub - K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock . E ) Quantification of D . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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D) Western blot analysis of Ub-K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock. E) Quantification of D. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


F ) Western blot analysis of Ub - K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock . G ) Quantification of F . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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F) Western blot analysis of Ub-K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock. G) Quantification of F. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


H ) Western blot analysis of Ub - K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock . I ) Quantification of H . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

H) Western blot analysis of Ub-K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock. I) Quantification of H. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


K ) Western blot of Ub - K48 proteins in the insoluble fraction of protein lysates of SOD1 - G93A mouse spinal cord plus or minus heat shock . L ) Quantification of panel K . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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K) Western blot of Ub-K48 proteins in the insoluble fraction of protein lysates of SOD1-G93A mouse spinal cord plus or minus heat shock. L) Quantification of panel K. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


M ) Chymotrypsin - like proteasome activity in spinal cord lysates from SOD1 - G93A mice . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) . Bordered points indicate biological replicates and un - bordered points indicate technical replicates . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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M) Chymotrypsin-like proteasome activity in spinal cord lysates from SOD1-G93A mice. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group). Bordered points indicate biological replicates and un-bordered points indicate technical replicates. Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


N ) Filter trap assay of protein lysates from the insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . O ) Quantification of panel N . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

N) Filter trap assay of protein lysates from the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. O) Quantification of panel N. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


P ) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . Scale bar equals 600nm . Q ) Quantification of P . ( n = 9 technical replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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P) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. Scale bar equals 600nm. Q) Quantification of P. (n=9 technical replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


( A ) Multiple sequence alignment of Atg8 homologues . Amino ‐ acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal _ W program . Underlined residues represent mutated amino acids in Atg8 .

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(A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8.


( B ) Alignment of Atg8 predicted three ‐ dimensional structure with its mammalian homologues . SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation . Orange represents the predicted Atg8 structure , and red , blue and green represent GATE ‐ 16 , GABARAP and LC3 , respectively .

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(B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively.


( C ) Three ‐ dimensional model of Atg8 . The predicted structure contains a ubiquitin fold decorated by two further amino ‐ terminal helices ( amino acids Tyr 49 , Leu 50 , Phe 77 and Phe 79 are marked in green ) .

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(C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).


( A ) Cells were grown to mid ‐ exponential phase and then incubated in the presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . Aminopeptidase I ( API ) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies ( upper panel ) .

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(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel).


( B ) Cells were pulse - labelled with [ 35S ] methionine and chased on non - radioactive starvation medium . Aliquots were taken at the indicated times and acid - soluble small peptides generated by proteolysis were determined .

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(B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined.


( C ) Cells were grown in control or in starvation medium and visualized by light microscopy . Arrows point to the pre ‐ autophagosomal structure ( PAS ) , and arrowheads point to GFP within the vacuoles .

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(C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles.


( D ) Cell extracts were subjected to SDS - polyacrylamide gel electrophoresis followed by immunoblot analysis using anti ‐ GFP antibodies . GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Atg8WT - HA , Atg8F77A - HA , Atg8F79A - HA or Atg8F77 / 79A - HA was transformed into Δatg8 cells . Cell extracts were prepared and subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) containing 6 M urea , followed by immunoblot analysis with anti - Atg8 antibodies .

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(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies.


( B ) Prediction of protein - protein interaction sites in Atg8 ( Neuvirth et al , 2004 ) . A three ‐ dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online ) with red representing the predicted protein - protein binding sites , and Phe 77 and Phe 79 are marked in green within the prediction site .

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(B) Prediction of protein-protein interaction sites in Atg8 ( Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online) with red representing the predicted protein-protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site.


( C ) Pull ‐ down assay of Atg8 mutants with Atg4 . His - Atg4 was attached to cobalt beads ( Talon metal ‐ affinity resin ) , washed and incubated with total cell extracts of Atg8 mutants . Elution was subjected to SDS - PAGE followed by western blot analysis using anti ‐ Atg8 and anti ‐ Atg4 antibodies .

(C) Pull‐down assay of Atg8 mutants with Atg4. His-Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS-PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies.


( D ) Localization of green fluorescent protein ( GFP ) - Atg8wt and GFP - Atg877 / 79 cells carrying Atg4 plasmid under normal ( control ) and starvation conditions . Arrows point to the pre ‐ autophagosomal structure , and arrowheads point to GFP within the vacuoles .

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(D) Localization of green fluorescent protein (GFP)-Atg8wt and GFP-Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles.


( E ) Aminopeptidase I ( API ) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti ‐ API antibodies . mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

(E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Cell extracts were subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis containing 6 M urea , followed by immunoblot analysis using anti ‐ Atg8 ‐ 15N peptide antibodies .

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(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies.


( B ) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy . Arrows point to the pre ‐ autophagosomal structure , whereas arrowheads point to GFP within the vacuoles .

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(B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles.


( C ) Aminopeptidase I ( API ) maturation and GFP cleavage were tested as described above . Asterisk represents nonspecific band . GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

(C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Δatg8 cells transformed with GFP - Atg8WT , GFP - Atg8L50A or GFP - Atg8Y49A constructs were grown to mid ‐ exponential phase and then incubated in the presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . API maturation was tested using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies .

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(A) Δatg8 cells transformed with GFP-Atg8WT, GFP-Atg8L50A or GFP-Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies.


( B ) Starvation ‐ induced protein degradation of Δatg8 harbouring the different mutants was tested as described above . API , aminopeptidase I ; GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(B) Starvation‐induced protein degradation of Δatg8 harbouring the different mutants was tested as described above. API, aminopeptidase I; GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) GFP - Atg8 localization was determined using fluorescence microscopy ( arrows depict pre ‐ autophagosomal structure , and arrowheads point to GFP within vacuoles ) . Cells were also tested for the appearance of autophagic bodies by using light microscopy .

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(A) GFP-Atg8 localization was determined using fluorescence microscopy (arrows depict pre‐autophagosomal structure, and arrowheads point to GFP within vacuoles). Cells were also tested for the appearance of autophagic bodies by using light microscopy.


( B ) Immunoblot analysis of different cell extracts was carried out using anti ‐ GFP antibodies ( asterisk represents nonspecific band ) .

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(B) Immunoblot analysis of different cell extracts was carried out using anti‐GFP antibodies (asterisk represents nonspecific band).


( C ) Atg8WT - HA , Atg8L50A - HA or Atg8Y49A - HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) followed by immunoblot analysis using anti ‐ HA antibodies ( lower panel ; asterisk represents nonspecific band ) .

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(C) Atg8WT-HA, Atg8L50A-HA or Atg8Y49A-HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band).


( D ) Atg8WT , Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h . Cell extracts were subjected to 13 . 5 % SDS - PAGE containing 6 M urea , followed by immunoblot analysis using anti ‐ Atg8 ‐ N15 antibodies . GFP , green fluorescent protein ; WT , wild type .

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(D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS-PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.


Overexpression of WT OGT , but not catalytic - inactive OGT mutant , increased the O - GlcNAc levels of TDP - 43 . OGT protein levels were examined by immunoblotting . Quantification of ( G ) .

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Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.   Quantification of (G).


Yeast cells expressing the indicated constructs were induced in galactose - containing medium , and representative images were shown using fluorescence microscopy to visualize TDP - 43 aggregates . DIC : Differential interference contrast . TDP - 43 aggregate foci numbers from individual cell of ( B ) were quantified from over 50 cells each of the indicated yeast strains . The foci numbers in individual cell > 3 were labelled with yellow , and the foci numbers in individual cell < 3 are labelled with blue .

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Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue.


SH - SY5Y cells treated with the indicated compounds were immunostained with TDP - 43 or pTDP - 43 and observed by immunofluorescence . Quantification of signal intensities of pTDP - 43 staining ( left ) or foci numbers per cell ( right ) . Cell number counted : left panel , " Mock " n = 58 , " GlcNAc " n = 35 , " EA " n = 45 , " GlcNAc + EA " n = 52 ; right panel , " Mock " n = 1167 , " GlcNAc " n = 1051 , " EA " n = 542 , " GlcNAc + EA " n = 815 .

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SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815.


SDS - soluble and insoluble fractions were isolated from SH - SY5Y cells expressing WT or catalytic - inactive mutant OGT treated with 20 μM EA , and the fractions were analyzed by immunoblotting . Quantification of ( F ) .

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SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting. Quantification of (F).


Cells expressing the indicated forms of GFP - TDP - 43 were subjected to immunoprecipitation , and the O - GlcNAcylated levels of TDP - 43 were examined . Quantification of ( G ) .

Cells expressing the indicated forms of GFP-TDP-43 were subjected to immunoprecipitation, and the O-GlcNAcylated levels of TDP-43 were examined.   Quantification of (G).


Walking speed of 10 - day old adult male flies of the indicated genotypes was calculated Mean ± SD . Unpaired two - tailed t - test , biological replicates : Control , n = 63 , WT , n = 45 , T199A , n = 70 , T233A , n = 87 ; 2TA , n = 43 .

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Walking speed of 10-day old adult male flies of the indicated genotypes was calculated Mean ± SD. Unpaired two-tailed t-test, biological replicates: Control, n = 63, WT, n = 45, T199A, n = 70, T233A, n = 87; 2TA, n = 43.


. Adult flies were fed with regular sucrose diet ( RS ) or high sucrose diet ( HS ) , and survival percentage of adult flies of the indicated genotypes were examined . Kaplan - Meier survival analysis , n ≥ 40 for each group .

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.Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.


CFTR splicing assay was performed in cells with knockdown of either OGT , TDP - 43 , or combination . ACTB levels served as loading controls ( top panel ) . The indicated protein levels were examined by immunoblotting ( bottom panel ) .

CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel).


CFTR splicing assay was examined in siTDP - 43 cells overexpressing the WT or mutant OGT by PCR analysis . ACTB levels served as loading controls ( top panel ) . Various protein levels were examined by immunoblotting ( bottom panel ) .

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CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel).


Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH - SY5Y cells with the indicated constructs ( top panel ) . TDP - 43 protein levels were examined by immunoblotting ( bottom panel ) .

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Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).


A BreastMark microarray platform ; Kaplan - Meier curves of overall survival ( OS ) , demonstrating that high expression of ESRP1 ( red line ) is associated with poor prognosis in ER + breast cancer . A log rank test was used to calculate P = 2 . 8 e - 07 ( n = 934 , number of events = 216 ) .

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A BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS), demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+ breast cancer. A log rank test was used to calculate P=2.8 e-07 (n= 934, number of events= 216).


B BreastMark microarray platform ; Kaplan - Meier curves of overall survival ( OS ) of ER - breast cancer . A log rank test was used to calculate P = 0 . 4481 ( n = 322 , number of events = 130 ) .

B BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS) of ER- breast cancer. A log rank test was used to calculate P=0.4481 (n= 322, number of events= 130).


C BreastMark microarray platform ; correlation of ESRP1 expression with overall survival in tamoxifen - treated patients . A log rank test was used to calculate P = 1 . 218 , e - 06 ( n = 210 , number of events = 49 ) .

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C BreastMark microarray platform; correlation of ESRP1 expression with overall survival in tamoxifen-treated patients. A log rank test was used to calculate P=1.218, e-06 (n= 210, number of events= 49).


D BreastMark microarray platform ; correlation of ESRP1 expression with overall survival in chemotherapy - treated patients . A log rank test was used to calculate the P = 0 . 28 ( n = 129 , number of events = 21 )

D BreastMark microarray platform; correlation of ESRP1 expression with overall survival in chemotherapy-treated patients. A log rank test was used to calculate the P=0.28 (n= 129, number of events= 21)


E The Cancer Genome Atlas Breast Invasive Carcinoma ( TCGA - BRCA ) RNA - seq dataset ; Kaplan - Meier curves of overall survival ( OS ) in ER + breast cancer , demonstrating that high expression of ESRP1 ( red line ) is associated with poor prognosis in ER + . A log rank test was used to calculate P = 0 . 00011 ( n = 656 , number of events = 62 ) .

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E The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER+ breast cancer, demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+. A log rank test was used to calculate P=0.00011 (n= 656, number of events= 62).


F The Cancer Genome Atlas Breast Invasive Carcinoma ( TCGA - BRCA ) RNA - seq dataset ; Kaplan - Meier curves of overall survival ( OS ) in ER - breast cancer . Red - high ESRP1 expression ; Black - low ESRP1 expression . A log rank test was used to calculate P = 0 . 19 ( n = 100 , number of events = 17 ) .

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F The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER- breast cancer. Red-high ESRP1 expression; Black-low ESRP1 expression. A log rank test was used to calculate P=0.19 (n=100, number of events=17).


A RT - qPCR confirmed the stable knockdown of ESRP1 expression in endocrine - resistant cells using a shRNA lentiviral system ( Mission TRC human shRNA constructs - Sigma ) ; 2 - control ( LCC2 cells transduced with pLKO . 1 control vector ) and 2C1 and 2C3 ( LCC2 cells transduced with pLKO . 1 shRNA ESRP1 ) . Clones were selected for expression verification ( clone 1 and clone 3 ) ; 9 - control ( LCC9 cells transduced with pLKO . 1 control vector ) , 9C2 and 9C3 ( LCC9 cells transduced with pLKO . 1 shRNA ESRP1 . Clones were selected for expression verification ( clone 2 and clone 3 ) . * represents P < 0 . 05 ; statistically significant . Data ( mean ± SD ) were calculated using two - way ANOVA based on three independent biological replicates .

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A RT-qPCR confirmed the stable knockdown of ESRP1 expression in endocrine-resistant cells using a shRNA lentiviral system (Mission TRC human shRNA constructs-Sigma); 2-control (LCC2 cells transduced with pLKO.1 control vector) and 2C1 and 2C3 (LCC2 cells transduced with pLKO.1 shRNA ESRP1). Clones were selected for expression verification (clone 1 and clone 3); 9-control (LCC9 cells transduced with pLKO.1 control vector), 9C2 and 9C3 (LCC9 cells transduced with pLKO.1 shRNA ESRP1. Clones were selected for expression verification (clone 2 and clone 3). * represents P <0.05; statistically significant. Data (mean ± SD) were calculated using two-way ANOVA based on three independent biological replicates.


B Western blot analysis using ESRP1 antibody GTX 131373 ( GeneTex ) . GAPDH is used as the reference control .

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B Western blot analysis using ESRP1 antibody GTX 131373 (GeneTex). GAPDH is used as the reference control.


C Relative cell density was determined by crystal violet assay . Cells were treated with TAM ( 10 - 6 M ) or ICI ( 10 - 9 M ) for 6 days in the presence and absence of E2 ( β - estradiol ; 10 - 10 M ) . Data ( mean + SD ) were calculated using two - way ANOVA based on three independent biological replicates .

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C Relative cell density was determined by crystal violet assay. Cells were treated with TAM (10-6 M) or ICI (10-9 M) for 6 days in the presence and absence of E2 (β-estradiol; 10-10 M). Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.