Figure 1.
Kinase inhibitor screen reveals signaling regulators of peroxisome abundance. (A) Western blot of WT and CRISPR/Cas9 PMP70-GFP HEK293T cells. Arrowheads indicate the size shift of the tagged PMP70-GFP. (B) Schematic of the peroxisome biogenesis regulators screen. CRISPR/Cas9 PMP70-GFP HEK293T cells were incubated in control or 1 μM of small molecules for 2 days. PEX3 overexpression was used as a negative control, and PEX19 overexpression was used as a positive control. Refer to Fig. S1, F and G, and Figs. S2 and S3 for the screen details. (C) Confocal microscopy of CRISPR/Cas9 PMP70-GFP HEK293T cells overexpressing PEX3-myc, flag-PEX19, or an empty vector. Images are included in Fig. S2 as controls. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100 cells pooled from three biological repeats, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (D–E) Confocal microscopy of peroxisomes in human primary fibroblasts AFF11 treated with indicated kinase inhibitors for 2 days (1 μM). Peroxisomes were visualized using PMP70 antibody, and nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 10 μm, *—abnormal nuclear morphology. (E) Quantification shows the number of peroxisomes per square micron of the cytoplasm in the flattened 3D image (indicated as cubic micron), mean ± SEM, N = 100 cells pooled from three biological repeats, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (F) Identified kinase inhibitors plotted on the human kinome network (Manning et al., 2002; Metz et al., 2018). Positive regulators (inhibition decreases the number of peroxisomes) are indicated in red, and negative regulators (inhibition increases the number of peroxisomes) are indicated in blue. PKC superfamily is shown in the inlet; G06983 inhibits indicated PKC isoforms. Source data are available for this figure: SourceData F1. OE, overexpressing.

Kinase inhibitor screen reveals signaling regulators of peroxisome abundance. (A) Western blot of WT and CRISPR/Cas9 PMP70-GFP HEK293T cells. Arrowheads indicate the size shift of the tagged PMP70-GFP. (B) Schematic of the peroxisome biogenesis regulators screen. CRISPR/Cas9 PMP70-GFP HEK293T cells were incubated in control or 1 μM of small molecules for 2 days. PEX3 overexpression was used as a negative control, and PEX19 overexpression was used as a positive control. Refer to Fig. S1, F and G, and Figs. S2 and S3 for the screen details. (C) Confocal microscopy of CRISPR/Cas9 PMP70-GFP HEK293T cells overexpressing PEX3-myc, flag-PEX19, or an empty vector. Images are included in Fig. S2 as controls. Quantification shows the number of peroxisomes per square micron of the cytoplasm in the 2D confocal image, mean ± SEM, N = 100 cells pooled from three biological repeats, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (D–E) Confocal microscopy of peroxisomes in human primary fibroblasts AFF11 treated with indicated kinase inhibitors for 2 days (1 μM). Peroxisomes were visualized using PMP70 antibody, and nuclei were stained with Hoechst (10 μg/ml). Representative images are shown; scale bar: 10 μm, *—abnormal nuclear morphology. (E) Quantification shows the number of peroxisomes per square micron of the cytoplasm in the flattened 3D image (indicated as cubic micron), mean ± SEM, N = 100 cells pooled from three biological repeats, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (F) Identified kinase inhibitors plotted on the human kinome network (Manning et al., 2002; Metz et al., 2018). Positive regulators (inhibition decreases the number of peroxisomes) are indicated in red, and negative regulators (inhibition increases the number of peroxisomes) are indicated in blue. PKC superfamily is shown in the inlet; G06983 inhibits indicated PKC isoforms. Source data are available for this figure: SourceData F1. OE, overexpressing.

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