Figure S4.
PPAR response and pexophagy during PKC regulation.(A) Confocal microscopy of peroxisomes in WT and PEX11b KO HEK293T overexpressing PKCδ-mCherry overexpressing (OE). Peroxisomes were stained with anti-PEX14 antibody. Scale bar: 10 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in PEX11b KO cells with and without overexpression of PKCδ-mCherry, mean ± SEM, N = 100 pooled from three biological repeats, ns—nonsignificant, Mann–Whitney. (B) Western blot of PPARα in HEK293T cells in control PMA (0.5 μM for 1 day) and Go6983 (5 μM for 2 days) conditions. Quantification shows the ratio of PPARα to loading control, mean ± SEM, N = 3, ***P < 0.001, *P < 0.05, one-way ANOVA. (C) Quantitative PCR of the peroxisomal genes in HEK293T cells in control and Go6983 5 μM conditions (2-day treatment). Quantification shows color coded relative expression levels calculated as a ΔCt (peroxisomal gene—GAPDH expression reference) and a ratio of Go6983/control expression, expressed as 2−ΔΔCt, mean ± SEM, N = 4, *P < 0.05, Kruskal–Wallis test. (D and E) Confocal microscopy of PPAR mCherry reporter in HEK293T cells expressing PPARα (D) and CHO (E) cells in control, Go6983 (5 μM for 2 days), or PMA conditions (0.5 μM for 1 day). Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown. Scale bar: 5 μm. Quantification shows average fluorescence intensity per cell, N = 50, ****P < 0.0001, Kruskal–Wallis test. (F) Western blot and confocal microscopy of NBR1 and control silencing in HEK293T CRISPR/Cas9 PMP70-GFP cells. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown. Scale bar: 5 μm; inlet: 1 μm. Quantification of the western blot shows the NBR1/GAPDH ratio quantified by the lane intensity on the western blot, mean ± SEM, N = 3, unpaired t test. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 150 pooled from three biological repeats, Mann–Whitney. (G) Western blot and confocal microscopy of NBR1/NIX and control silencing in HEK293T cells. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown. Scale bar: 5 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100 pooled from three biological repeats, **** - -<0.0001, one-way ANOVA. (H) Confocal microscopy of peroxisomes stained with anti-PMP70 antibody in WT and VAPB/VAPA KO HeLa cells in control and GFP-VAPB overexpression conditions. Source data are available for this figure: SourceData FS4.

PPAR response and pexophagy during PKC regulation. (A) Confocal microscopy of peroxisomes in WT and PEX11b KO HEK293T overexpressing PKCδ-mCherry overexpressing (OE). Peroxisomes were stained with anti-PEX14 antibody. Scale bar: 10 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in PEX11b KO cells with and without overexpression of PKCδ-mCherry, mean ± SEM, N = 100 pooled from three biological repeats, ns—nonsignificant, Mann–Whitney. (B) Western blot of PPARα in HEK293T cells in control PMA (0.5 μM for 1 day) and Go6983 (5 μM for 2 days) conditions. Quantification shows the ratio of PPARα to loading control, mean ± SEM, N = 3, ***P < 0.001, *P < 0.05, one-way ANOVA. (C) Quantitative PCR of the peroxisomal genes in HEK293T cells in control and Go6983 5 μM conditions (2-day treatment). Quantification shows color coded relative expression levels calculated as a ΔCt (peroxisomal gene—GAPDH expression reference) and a ratio of Go6983/control expression, expressed as 2−ΔΔCt, mean ± SEM, N = 4, *P < 0.05, Kruskal–Wallis test. (D and E) Confocal microscopy of PPAR mCherry reporter in HEK293T cells expressing PPARα (D) and CHO (E) cells in control, Go6983 (5 μM for 2 days), or PMA conditions (0.5 μM for 1 day). Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown. Scale bar: 5 μm. Quantification shows average fluorescence intensity per cell, N = 50, ****P < 0.0001, Kruskal–Wallis test. (F) Western blot and confocal microscopy of NBR1 and control silencing in HEK293T CRISPR/Cas9 PMP70-GFP cells. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown. Scale bar: 5 μm; inlet: 1 μm. Quantification of the western blot shows the NBR1/GAPDH ratio quantified by the lane intensity on the western blot, mean ± SEM, N = 3, unpaired t test. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 150 pooled from three biological repeats, Mann–Whitney. (G) Western blot and confocal microscopy of NBR1/NIX and control silencing in HEK293T cells. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown. Scale bar: 5 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100 pooled from three biological repeats, **** - -<0.0001, one-way ANOVA. (H) Confocal microscopy of peroxisomes stained with anti-PMP70 antibody in WT and VAPB/VAPA KO HeLa cells in control and GFP-VAPB overexpression conditions. Source data are available for this figure: SourceData FS4.

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