Fig. 1

Hypoxia (1% oxygen) suppresses AR expression and increases the CSC formation in RCC cells. A SW839 and OSRC-2 cells were exposed to hypoxia for 0 h, 12 h, 24 h, and 36 h. Real-time PCR was used to detect AR expression. B SW839 and OSRC-2 cells were exposed to hypoxia for 0 h, 12 h, 24 h, and 36 h. Western-blots were used to detect AR expression. C SW839 and SW839 with sh-AR1 and sh-AR2 were exposed to hypoxia (H) for 2 days, sphere formation assays were performed to evaluate the CSCs number. After 14 days incubation, colonies in five random fields per each well were counted under a microscope. D SW839 cells were virally transduced with sh-AR and cells exposed to hypoxia (H) or normoxia (N) for 2 days. Total RNAs were analyzed for CSCs markers including CD24, CD133, PAX2, CD105, SOX2 by real-time PCR. E OSRC-2 and ACHN cells were virally transduced with overexpressed AR and exposed to hypoxia (H) for two days, sphere formation assays were performed to evaluate the CSCs number. After 14 days incubation, colonies in five random fields per each well were counted under a microscope. F OSRC-2 cells were virally transduced with oe-AR and cells were exposed to hypoxia (H) or normoxia (N) for 2 days. Total RNAs were analyzed for CSCs markers including CD24, CD133, PAX2, CD105 and SOX2 by real-time PCR.(AR: androgen receptor; GAPDH: Recombinant protein of human glyceraldehyde-3-phosphate dehydrogenase; pLKO: pLKO.1-puro plasmid; sh-AR1/2: The plasmid encoding AR-shRNA1/2; pWPI: Addgene plasmid 12,254,; oe-AR: The plasmid encoding AR sequence, H:hypoxia; N: normoxia. * P<0.05, ** P<0.01, ***P<0.001)