Fig 2: Localization of VHL, HIF1A, HIF2A, VEGFA and P53 proteins in ccRCC and normal kidney. Immunoreactivity for (A and B) VHL, (C and D) HIF1A, (E and F) HIF2A, (G and H) VEGFA and (I and J) P53 proteins in normal kidney (A, C, E, G and I) and TNM stage 3 and Fuhrman grade 3 ccRCC sections (B, D, F, H and J) was demonstrated by immunohistochemical staining, as described in the Materials and methods. Scale bars, 10 µm (C-F) and 100 µm (A, B and G-J). Black and yellow arrows arrows indicate proximal and distal tubules of unchanged kidney morphological structure, respectively (A, C, E, G and I). ccRCC, clear cell renal cell carcinoma; VHL, Von Hippel-Lindau; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor.

Fig 3: Association between VEGFA and HIF2A protein expression, patient outcome and sunitinib treatment. Graphic presentation of multivariate regression analysis of the possible association between overexpression of the VEGFA and HIF2A proteins in tumor samples (independent variable), (A) overall or (B) progression-free survival and postoperative treatment with sunitinib. The variables were counted in non-parametric (0, 1) order, according to events (sunitinib usage or outcome) or VEGFA and HIF2A expression rates (high or low). Yellow-to-red areas represent increasingly significant associations between variables. HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor.

Fig 4: Expression analysis of the VHL, HIF1A, HIF2A, VEGFA and P53 genes in ccRCC at the protein level. Gene expression at protein level was assessed as described in the Materials and methods. (A) Comparison between tumor and normal kidney samples; (B) gene expression at protein level in tumor samples related to Fuhrman grade; and (C) gene expression at protein level in tumor samples related to TNM stage. Bars and whiskers represent mean ± standard deviation normalized to GAPDH level in each sample. *P<0.05, **P<0.01, ***P<0.001. (Wilcoxon signed-rank test for A, Mann-Whitney U test for B and C). ccRCC, clear cell renal cell carcinoma; VHL, Von Hippel-Lindau; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor; qPCR, quantitative polymerase chain reaction.

Fig 5: Kaplan-Meier's progression-free survival analysis of ccRCC patients related to clinicopathological and molecular data. (A) TNM classification. (B) Fuhrman grade. (C and D) VHL mRNA and protein expression, respectively. (E and F) HIF1A mRNA and protein levels, respectively. (G and H) HIF2A mRNA and protein expression, respectively. (I and J) VEGFA mRNA and protein levels, respectively. (K and L) P53 mRNA mRNA and protein levels, respectively. Cut-off values between increased and decreased expression rates were arbitrarily classified using median expression values of each gene/protein in control samples. Log-rank (Mantel-Cox) test was applied. ccRCC, clear cell renal cell carcinoma; VHL, Von Hippel-Lindau; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor.