( 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 .

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

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 .

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

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 .

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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 .

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

(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 .

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

(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 ) .

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

(G) 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 .

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

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 .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O

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 .

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

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 .

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

(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 .

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

(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 .

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

(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 .

O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O

(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 .

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

(B) 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 ) .

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

(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 .

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

(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 .

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

(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 ) .

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

(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 .

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

(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 .

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

(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 ) .

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

(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 .

O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) 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 .

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

(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 .

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

(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 ) .

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

(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 ) .

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

(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 ) .

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

(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 ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

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

(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).


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


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


by micro - CT of total volume per mouse in ( D ) CB - 839 - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , 20 post - infection . Vehicle - treated KP mice are 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 ) by micro - CT of total volume per mouse in , ( E ) AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , 20 post - infection . Vehicle - treated KP mice are 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 measured until week 20 . Relative response = average volume with treatment / average 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 measured until week 20 . Relative response = average volume with treatment / average 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 quantitation of total burden ( total / total lung ) in CB - 839 - , AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 20 post - infection . Vehicle - treated KP mice are same control group in both boxplots . 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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(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 KPΔNF1 tumors treated with vehicle , ( B ) CB - 839 , starting from day 12 measured until day 27 ( n = 6 tumors per per treatment ) Relative response = average volume with treatment / average 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 KPΔNF1 tumors treated with vehicle , C ) AOA starting from day 12 measured until day 27 ( n = 6 tumors per per treatment ) Relative response = average volume with treatment / average 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 KPΔNF1 tumors treated with vehicle , CB - 839 , or AOA ( D ) final masses of these tumors . or 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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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 KPΔNF1 tumors treated with vehicle , ( E ) CB - 839 starting from day 12 measured until day 27 ( n = 24 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 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 KPΔNF1 tumors treated with vehicle , ( F ) AOA starting from day 12 measured until day 27 ( n = 24 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 transduced with a luciferase vector .

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

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 volume with treatment / average 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 volume with treatment / average 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 - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( I ) CB - 839 starting from day 12 measured until day 30 ( n = 6 tumors per per treatment ) . Relative response = average volume with treatment / average 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 - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle ( J ) AOA starting from day 12 measured until day 30 ( n = 6 tumors per per treatment ) . Relative response = average volume with treatment / average 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 - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( K ) CB - 839 starting from day 12 measured until day 27 ( n = 12 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 transduced with a luciferase vector .

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

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

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

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 proteasome across nine tissues from ( n = 9 ) , ( n = 10 ) mice . Proportional distribution of trypsin - like activity of proteasome across nine tissues from ( n = 9 ) , ( 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 proteasome across nine tissues in mice . Each data point ( n = 9 n = 10 ) indicates data from a single mouse performed in triplicate . Each central error bar indicate mean + / - . * indicates p < 0 . 05 by unpaired two - students t - comparing 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 proteasome across nine tissues in mice . Each data point ( n = 9 n = 10 ) indicates data from a single mouse performed in triplicate . Each central error bar indicate mean + / - . * indicates p < 0 . 05 by unpaired two - students t - comparing 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 for K48Ub - linked in spinal cord ( A ) intestine ( C ) lysates from mice . ( B , D ) of panels A C , respectively . Values were normalized to actin as a loading control . Each central error bar indicate mean + / - of a biological replicates . Data information : * indicates p < 0 . 05 by unpaired two - students t - comparing 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 Ub - K48 linked in 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 ( CP ) or assembled with regulatory ( RP ) in ( n = 5 ) ( 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 ( CP ) or assembled with regulatory ( RP ) in ( n = 5 ) ( n = 5 ) spinal cord .

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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 ) of of assembled ( RP - CP ) versus unassembled ( CP ) proteasome in intestine of . Each central error bar indicate mean + / - of a biological replicates ( n = 5 per group ) . I ) of of RP - CP versus CP proteasome in spinal cord of . Each central error bar indicate mean + / - of a biological replicates ( n = 5 per group ) . Data information : * indicates p < 0 . 05 by unpaired two - students t - comparing 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 RP - CP / CP in ( purple ) ( blue ) intestine . K ) Correlation curve between chymotrypsin - like activity RP - CP / CP in ( purple ) ( 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 ( n = 9 ) ( n = 10 ) mice in young ( 3 - 5 months old ) or older ( 10 - 15 months old , ( n = 6 ) ( n = 7 ) ) groups . Each point indicates a single mouse . A ) Tissues that did not show significant decline , B ) Tissues that show significant decline in only , C ) Tissues that show significant decline in only or D ) in both . Each central error bar indicate mean + / - of a biological replicates ( n = 9 young , n = 6 older , n = 10 young , n = 7 older ) . P values obtained by unpaired two - students t - comparing young 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 of Ub - K48 in soluble fractions of intestine lysates from mice plus or minus heat shock . C ) of panel B . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing 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 of Ub - K48 in insoluble fraction of intestine lysates from mice plus or minus heat shock . E ) of D . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing 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 of Ub - K48 in soluble fractions of spinal cord lysates from mice plus or minus heat shock . G ) of F . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing 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 of Ub - K48 in insoluble fraction of spinal cord lysates from mice plus or minus heat shock . I ) of H . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing 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 in insoluble fraction of lysates of SOD1 - G93A mouse spinal cord plus or minus heat shock . L ) of panel K . Each central error bar indicate mean + / - of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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

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 error bar indicate mean + / - of a biological replicates ( n = 5 biological replicates per group ) . Bordered points indicate biological replicates un - bordered points indicate technical replicates . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing 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 of lysates from insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . O ) of panel N . Each central error bar indicate mean + / - of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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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 of in insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . Scale bar equals 600nm . Q ) of P . ( n = 9 technical replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing 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 alignment of Atg8 homologues . ‐ acid sequences of Atg8 its mammalian homologues are aligned using Clustal _ W program . Underlined residues represent mutated 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 PyMol was used for presentation . Orange represents predicted Atg8 structure , red , blue green represent GATE ‐ 16 , GABARAP 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 . predicted structure contains a ubiquitin fold decorated by two further ‐ terminal helices ( Tyr 49 , Leu 50 , Phe 77 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 ) were grown to mid ‐ exponential phase then incubated in presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . Aminopeptidase I ( API ) maturation was tested in incubated in control or starvation medium by western blot 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 ) were - labelled with [ 35S ] methionine chased on non - radioactive starvation medium . Aliquots were taken at indicated times 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 ) were grown in control or in starvation medium visualized by microscopy . Arrows point to pre ‐ autophagosomal structure ( PAS ) , arrowheads point to GFP within 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 ) extracts were subjected to SDS - polyacrylamide gel electrophoresis followed by immunoblot using anti ‐ GFP antibodies . GFP , green ; 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 . extracts were prepared subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) containing 6 M urea , followed by immunoblot 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.