Fig. 5

Mechanism dissection how AR/lncTCFL5-2/YBX1 signals may function via modulating SOX2 to alter the CSCs formation and chemotherapy resistance in RCC cells. A, B SW839 cells were virally transduced with sh-YBX1 or oe-YBX1 and then exposed to hypoxia and normoxia for 2 days. SOX2 expression was evaluated by western blot (A) and qPCR (B). C Bioinformatic analysis of potential YBX1 binding sites in SOX2 promoter. D Lysates of OSRC-2 cells were subjected to ChIP assay. ChIP products were amplified by PCR reactions. E, F Co-transfection of SOX2 promoter constructs containing the wild type or mutant region into OSRC-2 cells and luciferase assay was applied to detect the luciferase activity. G OSRC-2 cells were lentivirally transduced with pLVTHM or oe-lncTCFL5-2 or co-transfected with oe-lncTCFL5-2 and sh-SOX2. Total RNAs were analyzed for CSCs markers CD24, CD133, SOX2, NANOG, and CD105, PAX2, by real-time PCR. H SW839 and OSRC-2 cells were lentivirally transduced with pLVTHM or oe-lncTCFL5-2 or co-transfected with oe-lncTCFL5-2 and sh-SOX2. Western blot analysis of the expressions of CD24, CD133, SOX2. I SW839 and OSRC-2 cells were lentivirally transduced with pLVTHM or oe-lncTCFL5-2 or co-transfected with oe-lncTCFL5-2 and sh-SOX2. Sphere formation assays were performed to evaluate the CSC phenotype. After 14 days of incubation, colonies in five random fields per each well were counted under a microscope. J MTT assay was used to determine viability of cells exposed to hypoxia comparing to normoxia in SW839 cells with sh-lncTCFL5-2 or without sh-lncTCFL5-2 with Sunitinib treatment. K SW839 and OSRC-2 cells were exposed to hypoxia and normoxia for 2 days, and then treated with THZ1. Total RNAs were analyzed for lncTCFL5-2 by real-time PCR. L SW839 cells were lentivirally transduced with or without sh-lncTCFL5-2, then exposed to hypoxia and normoxia, treated with DMSO or Sunitinib combined with CDK-7 inhibitor. MTT assay was used to detect the cell viability. M OSRC-2 cells were virally transduced with pWPI or wildtype or mutant lncTCFL5-2, then treated with different concentrations of THZ1 followed by MTT assay