Signaling regulators of peroxisome abundance. (A) Schematic of the genomic DNA region corresponding to the end of the human PMP70 open reading frame endogenously tagged with -GFP-polyA-Blasticidin. (B and C) Confocal microscopy of the peroxisomal import marker mCherry*4-SKL expressed in HEK293T PMP70-GFP, and (B) confirmation of the PMP70-GFP peroxisomal localization. Fluorescence intensity profiles through single peroxisomes are shown. Scale bar: 5 μm. (D and E) Comparison of WT and PMP70-GFP–tagged HEK293T. Confocal images and quantification of perimeter (N = 1,000), area (N = 1,000), and density of peroxisomes (N = 100) are shown, mean ± SEM, Mann–Whitney. (F and G) Quantification of the number of peroxisomes per square micron of the cytoplasm in the HEK293T cells treated with 1 μM of the indicated small molecule, mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (G)Significant screen hits are plotted, and (F) complete screen. Refer to Figs. S2 and S3 for the confocal images, N = 100 for each molecule. (H) Confocal microscopy of HEK293T CRISPR/Cas9 PMP70-GFP cells overexpressing (OE) PKCα-mCherry, PKCδ-mCherry, or PKCζ-mCherry. Scale bar: 5 μm. See zoomed in images in Fig. 2 B. (I) Western blot of PKCδ in WT and CRISPR/Cas9 KO HEK293T cells. Confocal microscopy (PeroxiSPY555 0.5 μM 10 min), and 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, ns—nonsignificant, Mann–Whitney. Representative images are shown. Scale bar: 5 μm; inlet: 1 μm. Source data are available for this figure: SourceData FS1.