Fig. 4

A fraction of PPFIA4 localizes in the mitochondria and confers increased mitochondrial function. A. KEGG pathway gene set enrichment analysis of downregulated genes in C4-2B cells with PPFIA4 knockdown. C4-2B cells were transfected with siNC or siPPFIA4 for 48 hours. Total RNA was then extracted and used to perform RNA-Seq analyses. KEGG pathway gene set enrichment analysis was carried out to examine the pathway enrichment. B. Organelle localization analysis of PPFIA4 interacting proteins by Web Gestalt (http://www.webgestalt.org/). C4-2B cell lysates were immunoprecipitated with PPFIA4 antibody and resolved by SDS–PAGE. Liquid chromatography-tandem mass spectrometry analysis was performed to identify its interacting proteins. C. Localization of PPFIA4 (green) in LNCaP cell mitochondria (red) was verified by immunofluorescent staining with or without androgen deprivation. Representative images are shown with a 10-μm scale bar. Magnified images from the regions marked by rectangles in the top panel was showed in the bottom panel. Mito, mitochondria. FBS, fatal bovine serum. CSS, charcoal-stripped serum. D. The protein levels of PPFIA4 in total cell lysates (Total), cytosolic fraction (Cyto), and mitochondrial fraction (Mito) were analyzed by western blotting in LNCaP cells with or without androgen deprivation. COXIV and tubulin were used as mitochondrial and cytosolic markers. E-F. Measurement of OCR in indicated PCa cells with PPFIA4 overexpression or knockdown and treated with or without androgen deprivation and enzalutamide (10 μM). Representative recording of OCR during extracellular flow analysis (“Seahorse”) is shown in the up panel, and quantitative analysis of the calculated basal and maximum respiratory rates, ATP production rate and spare respiratory capacity are shown in the bottom panel. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. m, minutes. FBS, fatal bovine serum. CSS, charcoal-stripped serum. ENZ, enzalutamide. G-L. Measurement of membrane potentials, NADPH/ NADP⁺ ratio, and ROS levels in LNCaP (G-I) and C4-2B (J-L) cells with PPFIA4 overexpression or knockdown and treated with or without androgen deprivation. **p < 0.01, ***p < 0.001, ***p < 0.001, ****p < 0.0001. FBS, fatal bovine serum. CSS, charcoal-stripped serum. M. Cell proliferation was measured by cell counts in C4-2B cells with PPFIA4 knockdown and treated with or without ROS scavenger NAC (5 nM). Cell number was counted at the indicated time points and all the numbers were normalized to day 0. All error bars represent the SD of at least three replicates from three independent experiments. **p < 0.01, ***p < 0.001. d, days