Fig. 6

PFKFB4 phosphorylates NCOA3 in ccRCC. Phosphoproteomic assay using Tandem Mass Tag (TMT) technique in 786O cells with PFKFB4-knockdown (KD) versus negative control (NC) showing A) Clustering heatmap of deferentially phosphorylated peptides; B) Venn diagram of showing common proteins both significantly phosphorylated in phosphoproteomics and shown to interact with PFKFB4 in mass spectrum with C) validation using co-immunoprecipitation assay in lentiviral PFKFB4-overexpressed 786O cells; D) Violin plots of phosphorylation at each site of NCOA3 detected in the phosphoproteomics, Student’s t-test; E) Promoter luciferase assay showing activity of NCOA3 in 786O cells with PFKFB4-KD or NC, Student’s t-test; F) Western blotting showing phosphorylation levels at 2 sites of NCOA3 by different doses of adenoviral (Av) PFKFB4 overexpression and G) ratio of Phospho-NCOA3 over total NCOA3 measured by densitometry, two-way ANOVA; H) mRNA level of NCOA3 detected by q-PCR in 786O cells with PFKFB4-KD (2 shRNAs) or NC, one-way ANOVA; I) Reproduced from the Cancer Genome Atlas (TCGA) clear-cell renal cell carcinoma (KIRC) dataset, shown was expression correlation between PFKFB4 and NCOA3 at RNA-seq platform, both Pearson and Spearman correlations listed; J) Representative immunohistochemical staining of PFKFB4 and NCOA3 in the same ccRCC sample, bar = 200 μm. (All in vitro assays performed in triplicates and at least 3 biological replicates; ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)