EMBO/SourceData huggingface dataset:
Figure 8(A) Cellular protein extracts were isolated from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot.(B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.
Figure 9(A) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient-derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified, and normalized to control fibroblasts. Data are expressed as a mean ± SEM (n=3, *p=0.01).(B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]).(C) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]).(D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.(E) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot.(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.
Figure 1(A) IDH1 acetylation levels upon treatment with NAM or TSA. Flag-tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period.(B) Five putative lysine residues were mutated. Acetylation levels of Flag-bead-purified IDH1 were determined by Western blot analysis using a pan-anti-acetyllysine antibody. Relative IDH1 acetylation ratios were calculated after normalizing against Flag.(C) Mutation of IDH1 K224R and K224Q resulted in altered acetylation levels using Western Blot.(D) IDH1 catalytic activity of IDH1 K224 mutants in vitro. Wild-type IDH1 and K224R, K224Q mutants were expressed in HEK293 cells. Proteins were purified by immunoprecipitation (IP), IDH1 levels were normalized for protein, and activity assays were performed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For multiple comparisons in (D), a one-way ANOVA with Newman-Keuls post hoc test was used to test for statistical significance.(E-F) Steady-state kinetic analysis of IDH1 and variants in HEK293T cells. Comparison of IDH1 WT and K224R and K224Q showed that the K224 site was important in catalysis. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For (E-F), statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.(G) IDH1 directly interacted with SIRT2 but not SIRT1. SIRT2 deacetylated IDH1 effectively. Flag tagged IDH1 and HA tagged SIRT1 or SIRT2 were co-transfected into cells. The acetylation level of Flag-bead-purified IDH1 and the protein association between IDH1 and SIRT1 or SIRT2 were determined by Western blot.(H) Co-immunoprecipitation showed endogenous proteins of IDH1 and SIRT2 interacted with each other.(I) SIRT2 interacted with IDH1 in vitro. GST pull-down assay with GST-fused SIRT2 and Flag-tagged IDH1 proteins.(J) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot analysis.(K) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity. SIRT2 H187Y was a catalytically inactive mutant.
Figure 2(A) Colony formation ability after transfection of SIRT2 plasmids, AGK2 and SIRT2 siRNA. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.(B) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2. Stained cells in the lower chambers were quantified. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.(C) Cell migration ability was assessed by wound healing assay in HCT116 cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure C, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.(D) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids, SIRT2 siRNA or AGK2. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.(E) Immunohistochemical analysis of SIRT2 protein in representative colorectal tissue specimens is shown (magnification ×100). Weak cytoplasmic staining for SIRT2 was observed in CRC tissues and metastatic tissues, whereas strong cytoplasmic staining was observed in noncancerous tissues.
Figure 4(A) Gene expression profile array analysis in HCT116 cells. Representative genes with higher fold difference (up or down) compared IDH1 K224R cells with IDH1 WT cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition).(B) ChIP assays for HIF1α and its binding motif. Antibodies anti-IgG and anti- HIF1α were used in the ChIP assays. qRT-PCR was performed to quantify the binding activity. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B , statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant.(C-D) The transcriptional activity of the SRC promoter was greatly increased after co-expression of HIF1α and SRC reporter in wild type. SRC promoter constructs containing a potential HIF1α binding motif (-1636~-1629 bp and -1466~-1459 bp). Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant.(E) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy-HIF1α. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, HIF1α and hydroxy-HIF1α was measured by Western blot assay.(F) HCT116 cells of IDH1 K224Q were cotreated with Octyl-α-KG and NAC, the expression of HIF1α and SRC was further evaluated.HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For G statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for lactate levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.
Figure 5(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 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) 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: 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) 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.
Figure 6(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 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.
Figure 7(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) 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.
Figure 1A. 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.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. 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 algorithmE. 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.
Figure 2A. 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), 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 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 and mutant animals. Comparisons were performed with the WT.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 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.
Figure 3B. 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 and mutant worms.
Figure 4A. 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 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 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 and osm-3 mutant animals.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.
Figure 1(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.(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.
Figure 2(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).
Figure 3(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.(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.(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).
Figure 4Rathippocampal 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.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.
Figure 5(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.(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.
Figure 6(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.