Fig 1: CLUH promotes mitophagy Western blot analysis of total protein extracts from livers of 8-week-old Li-Cluh WT and Li-Cluh KO mice probed with indicated antibodies. GAPDH was used as loading control.Quantification of Western blot shown in (A). WT: n = 4; KO: n = 3.Western blot analysis of isolated mitochondria from Li-Cluh WT and Li-Cluh KO mice probed with indicated antibodies. Ponceau S staining was used as a loading control.Quantification of Western blot shown in (C) (n = 3 mice per genotype).Confocal images of liver cryosections derived from Li-Cluh WT and Li-Cluh KO-LC3-GFP mice and stained with anti-TOM20 antibody. On the right side, indicated boxes were 3.5× magnified. Arrows point to colocalizing spots. Scale bar, 25 µm.Manders’ colocalization coefficient between TOM20 and LC3 from experiments shown in (E). Five images were analyzed per animal to get an averaged value (n = 5 mice per genotype).Confocal images of primary hepatocytes derived from Li-Cluh WT and Li-Cluh KO-LC3-GFP mice and stained with anti-TOM20 antibody. When indicated, cells were treated with rapamycin (200 nM for 4 h). Bottom panels show 2.3× magnified areas for each channel of indicated boxes. Arrows point to colocalizing spots. Scale bar, 10 µm.Manders’ colocalization coefficient from experiment shown in (G) (n = 50 cells isolated from 3 mice per genotype).Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101–152 and grown for 16 h in galactose medium or in medium containing 10 µM antimycin A/oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 µm.Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n = 3–5 independent experiments, > 100 cells per experiment).Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101–152, incubated with 200 nM rapamycin, and grown for 16 h in galactose medium or in medium containing 10 µM antimycin A/oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 µm.Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n = 3–5 independent experiments, > 100 cells per experiment).Data information: In (B, D, F, J, L), data are presented as histograms showing the mean ± SEM. In (H), data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values. (B, D, F) ***P = 0.001; *P = 0.05 (Student's t-test). (H, J, L) *P = 0.05; **P = 0.01; ***P = 0.001 (one-way ANOVA, Tukey's multiple comparison test).Source data are available online for this figure.
Fig 2: Overexpressed CLUH forms CHX resistant granules in HeLa cells AConfocal images of WT and CLUH KO HeLa cells stained with anti-CLUH antibody. Scale bar, 10 µm.BConfocal images of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH (marked with asterisks). These images were overexposed to detect cells with CLUH expression at endogenous level CLUH. Asterisks indicate overexpressing cells. Scale bar, 10 µm.C, DConfocal images of HeLa cells overexpressing untagged CLUH (C) or FLAG-tagged CLUH (D) stained with indicated antibodies. Scale bar, 10 µm.EConfocal images of HeLa cells overexpressing untagged CLUH treated with or without CHX and stained with the indicated antibodies. Scale bar, 10 µm.FQuantification of percentage of cells with CLUH granules of experiment shown in (E) (n = 3 independent experiments, > 50 cells per condition per replicate).GConfocal images of HeLa cells treated with arsenite with or without CHX and stained with anti-G3BP1 antibody. Scale bar, 10 µm.HQuantification of percentage of cells with G3BP1 granules of experiment shown in (G) (n = 3 independent experiments, > 50 cells per condition per replicate).ILive imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and treated with arsenite with and without CHX. Cells were recorded for a maximum of 30 min. Scale bar, 10 µm.JTotal number of cells analyzed by live imaging for the indicated experiments. “Positive” indicates a cell which forms G3BP1 granules at the end of the recording.Data information: In (F, H), data are presented as histograms showing the mean ± SEM. (H) ***P = 0.001 (Student's t-test).
Fig 3: Transcriptomic and proteomic profile of WT and KO hepatocytes A, BVolcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal (A) and HBSS (B) conditions.C2D score plots of enriched pathways in transcriptomics analysis of KO and WT hepatocytes under basal condition and upon HBSS (2 h).D2D score plots of enriched pathways in proteomics analysis of KO and WT hepatocytes under basal condition and upon HBSS (2 h).E2D score plots of enriched pathways in proteomics versus transcriptomics analysis of KO and WT hepatocytes under basal condition.FCorrelation of protein fold changes in KO with respect to WT hepatocytes under basal condition and upon HBSS (2 h). Only genes previously found in CLUH RIP experiments in HeLa cells (Gao et al, 2014) are plotted. Inset shows magnification of upper part of the graph.
Fig 4: G3BP1 granules colocalize with LAMP1 A, BConfocal images of primary hepatocytes isolated from Li-Cluh WT (A) and Li-Cluh KO (B) mice and stained with anti-G3BP1 and anti-LAMP1 antibodies. 7× magnified areas are shown on the right side. Scale bar, 10 µm.CManders’ colocalization coefficient between G3BP1 and LAMP1 signals of experiments shown in (A, B) (n = 35 cells with granules from 3 mice per genotype).Data information: In (C), data show the mean ± SEM. (C) *P = 0.05 (Student's t-test); ***P = 0.001 for time; and **P = 0.01 for interaction time–genotype (two-way ANOVA).
Fig 5: CLUH forms granules upon starvation AConfocal images of liver cryosections and primary hepatocytes of Li-Cluh WT and Li-Cluh KO mice stained with anti-CLUH antibody. Scale bar, 10 µm.BConfocal images of liver cryosections of fed and starved Li-Cluh WT and Li-Cluh KO mice stained with anti-CLUH antibody. Right panels show 6.5× magnified boxed areas. Scale bar, 10 µm.CConfocal images of primary hepatocytes cultured in indicated media and stained with anti-CLUH antibody. Right panels show 4.5× magnified boxed areas. Scale bar, 10 µm.D, EConfocal images of primary hepatocytes cultured in indicated media and stained with (D) anti-G3BP1 or (E) anti-DCP1A and anti-CLUH antibodies. Right panel shows 5× enlargement of indicated area. Scale bar, 10 µm.
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