Figure 3.
PKC regulation depends on peroxisome division. (A) Schematic of PEX19 complementation assay. (B–D) Confocal microscopy of the peroxisomes in HEK293T WT, PEX19 KO, and PEX19 KO cells overexpressing flag-GFP-PEX19 in control, Go6983 1 μM, and PMA 0.5 μM conditions. Cells were fixed and visualized on identified days. (B) Quantification shows the ratio of cells that restored peroxisomes among the transfected cells. Scale bar: 5 μm. (C) Western blot of PMP70 expression on day 4 of the complementation assay. (E) Confocal microscopy of peroxisomes in HEK293T CRISPR/Cas9 flag-GFP-PEX11b cells treated with 0.5 μM PMA for 2 h. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 5 μm; inlet: 1 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100, ****P < 0.0001, Mann–Whitney test. (F) Western blot of PEX11b and PMP70 in the WT and CRISPR/Cas9 PMP70-GFP PEX11b KO HEK293T cells. Arrowheads indicate the size shift of the tagged PMP70-GFP. (G and H) Confocal microscopy of peroxisomes in HEK293T CRISPR/Cas9 PMP70-GFP WT and PEX11b KO cells grown in control or Go6983 5 μM conditions. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 5 μm; inlet: 1 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100 cells pooled from 3 biological repeats, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (I) Confocal microscopy of peroxisomes in HEK293T CRISPR/Cas9 MFF KO cells grown in control or Go6983 5 μM conditions. Western blot shows MFF in WT and MFF KO cells. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 5 μm; inlet: 1 μm. (J) Quantification of the length of the extended peroxisomes in HEK293T CRISPR/Cas9 MFF KO cells grown in control or Go6983 5 μM conditions, mean ± SEM, ****P < 0.0001, Mann–Whitney test. Source data are available for this figure: SourceData F3. ns, nonsignificant.

PKC regulation depends on peroxisome division. (A) Schematic of PEX19 complementation assay. (B–D) Confocal microscopy of the peroxisomes in HEK293T WT, PEX19 KO, and PEX19 KO cells overexpressing flag-GFP-PEX19 in control, Go6983 1 μM, and PMA 0.5 μM conditions. Cells were fixed and visualized on identified days. (B) Quantification shows the ratio of cells that restored peroxisomes among the transfected cells. Scale bar: 5 μm. (C) Western blot of PMP70 expression on day 4 of the complementation assay. (E) Confocal microscopy of peroxisomes in HEK293T CRISPR/Cas9 flag-GFP-PEX11b cells treated with 0.5 μM PMA for 2 h. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 5 μm; inlet: 1 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100, ****P < 0.0001, Mann–Whitney test. (F) Western blot of PEX11b and PMP70 in the WT and CRISPR/Cas9 PMP70-GFP PEX11b KO HEK293T cells. Arrowheads indicate the size shift of the tagged PMP70-GFP. (G and H) Confocal microscopy of peroxisomes in HEK293T CRISPR/Cas9 PMP70-GFP WT and PEX11b KO cells grown in control or Go6983 5 μM conditions. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 5 μm; inlet: 1 μm. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100 cells pooled from 3 biological repeats, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (I) Confocal microscopy of peroxisomes in HEK293T CRISPR/Cas9 MFF KO cells grown in control or Go6983 5 μM conditions. Western blot shows MFF in WT and MFF KO cells. Nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 5 μm; inlet: 1 μm. (J) Quantification of the length of the extended peroxisomes in HEK293T CRISPR/Cas9 MFF KO cells grown in control or Go6983 5 μM conditions, mean ± SEM, ****P < 0.0001, Mann–Whitney test. Source data are available for this figure: SourceData F3. ns, nonsignificant.

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