FIG. 6.

HIF2α downregulation in 786-O cells producing type 2B mutants results in decreased tumorigenicity in vivo. (A) Immunoblot (IB) analysis of 786-O cells producing the indicated pVHL variants (or infected with empty vector) and subsequently infected with retroviruses encoding shRNAs targeting HIF2α [shHIF2α(#2) and shHIF2α(#3)] or luciferase (shLuc). (B) Optical density (OD) at 450 nm, which reflects viable cell number, of 786-O cells studied in panel A and maintained in 10% fetal bovine serum. Standard deviation is <0.2 for all data points. (C) BrdU incorporation of 786-O cells stably infected with retroviruses as in panel A and maintained in serum-free DMEM supplemented with 1% insulin-transferrin-selenium (1% ITS) for 72 h prior to BrdU pulse for 2 h. Error bars indicate standard deviations. (D and E) Masses of tumors formed by 786-O cells stably infected with retroviruses as in panel A 8 weeks after subcutaneous injection in nude mice. Each mouse was injected with cells expressing luciferase shRNA or HIF2α shRNA on opposite flanks. The number of tumors analyzed is indicated in parentheses. Error bars indicate 1 standard error of the mean (P < 0.05). (F) Calculated masses of tumors formed by 786-O cells stably infected with a retrovirus encoding pVHL(Y112N) and the indicated shRNA retrovirus 10 weeks after intraparenchymal renal injection in nude mice. The presence of tumor cells was confirmed by hematoxylin and eosin staining. The tumor mass was calculated by subtracting the mass of the left kidney (normal) from the mass of the right kidney (implanted with tumor). Numbers in parentheses indicate the number of animals with tumors divided by the number of animals injected. Error bars indicate 1 standard error of the mean (P < 0.05).