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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: PPFIA4 promotes castration-resistant prostate cancer by enhancing mitochondrial metabolism through MTHFD2

Fig. 5

PPFIA4 interacts with MTHFD2 in mitochondria and accentuates mitochondrial function via MTHFD2. A. Identification of proteins that interact with PPFIA4 by Co-IP assays and mass spectrometry analysis. C4-2B cell lysates were immunoprecipitated with PPFIA4 antibody and subjected to SDS–PAGE. Coomassie blue staining were then performed, protein bands were cut and subjected to mass spectrometry. B-C. The binding potential between PPFIA4 and MTHFD2 in the whole cell lysates (Total, B), cytoplasmic fraction (Cyto) and mitochondrial fraction (Mito) were performed by Co-IP assays in C4-2B cells. IgG serves as negative control. COXIV and tubulin were used as mitochondrial and cytosolic markers. D. The binding potential between PPFIA4 and MTHFD2 was analyzed by Co-IP assays in LNCaP cells with or without androgen deprivation. IgG serves as negative control. E. The distribution of PPFIA4 (green) and MTHFD2 (red) was analyzed by immunofluorescence staining in LNCaP cells with or without androgen deprivation. Representative images are shown with a 10-μm scale bar. FBS, fatal bovine serum. CSS, charcoal-stripped serum. F. Measurement of OCR in C4-2B cells transfected with PPFIA4 overexpression plasmid for 24 hours and subsequent MTHFD2 siRNA for another 24 hours. Representative recordings of OCR during extracellular flow analysis (“Seahorse”) are 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 < 0.001. m, minutes. G-H. Measurement of NADPH/NADP+ ratio (G) and ROS levels (H) in C4-2B cells transfected with PPFIA4 overexpression plasmid for 24 hours and subsequent MTHFD2 siRNA for another 24 hours. *p < 0.05, **p < 0.01. I-K. Cell counts (I), cell clone formation assays (J), and cell apoptosis assays (K) were performed in C4-2B cells co-transfected with PPFIA4 overexpression plasmid for 24 hours and subsequent MTHFD2 siRNA for another 24 hours. *p < 0.05, **p < 0.01, ***p < 0.001. d, days

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