Fig. 2
Hypoxia-suppressed AR expression may alter the expression of lncRNAs in RCC cells. A SW839 cells, pre-cultured in the hypoxia condition (1% oxygen level for 24Â h) and normoxia condition. Volcano plots of the differentially expressed lncRNAs: red spots represent fold change more than 0.6 and P valve less than 0.05. B Western-blot showed the efficiency of sh-AR in SW839 cells and overexpressed AR (oe-AR) in OSRC-2 cells. C The list of top 20 upregulated lncRNAs by hypoxia. D SW839 and OSRC-2 cells were virally transduced with sh-AR or oe-AR, and then cells were exposed to hypoxia (H) or normoxia (N) for 2Â days. Total RNAs were analyzed by Q-PCR to show these 20 lncRNAs expression. The results showed that three lncRNAs (lnc-CTB-114C7.4, lnc-TCFL5-2(XLOC_013838)and LINC00312) were suppressed by AR. E SW839 cells were virally transduced with pLVTHM, sh-lnc-CTB-114C7.4, sh-lncTCFL5-2, sh-LINC00312 and cells were exposed to hypoxia (H) for 2Â days. Sphere formation assay were performed to evaluate the CSCs numbers. After 14Â days of incubation, colonies in five random fields per each well were counted under a microscope. F SW839 and OSRC-2 cells were virally transduced with sh-lncTCFL5-2 and then cells were exposed to hypoxia and normoxia for 2Â days. Total RNAs were analyzed for CSCs markers CD24, CD133, PAX2, CD105, SOX2 by real-time PCR. G OSRC-2 cells were virally transduced with oe-lncTCFL5-2 and then cells exposed to hypoxia (H) for 2Â days. Sphere formation assay were performed to evaluate the CSCs numbers. H SW839 and OSRC-2 cells were virally transduced with oe-lncTCFL5-2 and then cells exposed to hypoxia and normoxia for 2Â days. Total RNA were analyzed for CSCs markers CD24, CD133, PAX2, CD105, SOX2 by real-time PCR. I ACHN and OSRC-2 cell were virally transduced with oe-AR or were co-transfected with oe-AR and oe-lncTCFL5-2, and then cells exposed to hypoxia for 2Â days. Sphere formation assays were performed to evaluate the CSCs phenotype. (AR: androgen receptor; pLVTHM: Addgene plasmid 12,247; sh-lnc-CTB-114C7.4: The pLVTHM plasmid encoding lnc-CTB-114C7.4 shRNA, sh-lncTCFL5-2: The pLVTHM plasmid encoding lncTCFL5-2 shRNA, sh-LINC00312: The pLVTHM plasmid encoding LINC00312 shRNA; oe-AR: The plasmid encoding AR sequence; oe-lncTCFL5-2: The plasmid encoding lncTCFL5-2 sequence.)