Figure 2. VHL-HIF Signal Output Modulation Associated with ccRCC Progression.

a. Effects of VHL reintroduction on CXCR4 expression measured by quantitative RT-PCR analysis. Expression normalized to 786-O cells transduced with the control vector.

b. Kaplan-Meier analysis of metastasis-free survival in patients with ccRCC. Classification based on CXCR4 mRNA expression in primary ccRCCs, top 1/3 shown in red, bottom 2/3 in blue. P-value from a Cox proportional hazards model with CXCR4 expression treated as a continuous variable. n = 91.

c. Effects of VHL reintroduction on VEGFA and ADM expression measured by quantitative RT-PCR analysis. Expression normalized to 786-O cells transduced with the control vector.

d. Venn-diagram showing the overlap between the 155 metastasis-associated genes and genes changed after VHL reintroduction into the 786-M1A cells. Statistical significance of overlap tested by Fisher’s exact test.

e. Effects of VHL reintroduction on gene expression measured by quantitative RT-PCR analysis. Expression normalized to 786-O cells transduced with the control vector.

f. Effects of HIF2α knockdown on gene expression measured by quantitative RT-PCR analysis in 786-M1A cells. Expression normalized to the cells transduced with the hairpin control vector.

g. Effects of VHL reintroduction on gene expression measured by quantitative RT-PCR analysis. Expression normalized to RFX-631 cells transduced with the control vector.

h. Effects of VHL reintroduction on gene expression measured by quantitative RT-PCR analysis. Expression normalized to OS-LM1 cells transduced with the control vector.

i. A schematic summarizing the hypothesis that when VHL loss stabilizes HIF, early ccRCC genes such as VEGF and ADM are highly expressed whereas the activation of pro-metastatic HIF target genes such as CXCR4 and others requires additional amplifying mechanisms. Error bars represent the 95% confidence interval based on multiple PCR reactions in all panels.