Fig. 4

PFKFB4 is transcribed by HIF-1α in ccRCC. A) Reproduced from Cistrome ChIP-Seq dataset, shown were predicted transcription factors that could bind promoter of PFKFB4 in kidney tissue; B) Relative firefly-luciferase activity of PFKFB4 in 786O cells where Firefly luciferase activity was normalized to Renilla luciferase activity for all samples to yield relative luciferase activity, Student’s t-test; C) Reproduced from the Cancer Genome Atlas (TCGA) clear-cell renal cell carcinoma (KIRC) dataset, shown was expression correlation between PFKFB4 and HIF1A in ccRCC samples measure in microarray platform, Pearson correlation test; D) ChIP-PCR analysis of Flag marks (HIF1A) at the PFKFB4 promoter region in 786O cells with schematic diagram of PFKFB4 promoter regions and mouse IgG serving as negative control; E) HIF-1α binding to HRE-D (-270 ~ -290) site in the PFKFB4 promoter under the hypoxic condition in 786O cells as determined by ChIP assays under the hypoxic or normoxic conditions for 36 h before assays, with amount of DNA fragments pulled-down determined by real-time PCR; F) Western blotting showing HIF-1α and PFKFB4 level in 4 ccRCC cell lines with different basal HIF-1α level with knockdown (sh) or adenoviral overexpression (Av) of HIF1A; Reproduced from TCGA-KIRC dataset, shown were G) Genomic alteration of 3p and location of PFKFB4 in relation to VHL in ccRCC; H) Oncoprint of genetic alterations of HIF1A and PFKFB4 with mutual exclusivity detected by Chi-square test; I) mRNA expression of PFKFB4 against its copy number in ccRCC, Student’s t-test. (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)