Fig. 4

Mechanism dissection how AR-suppressed lncTCFL5-2 may increase CSCs the phenotype via interacting with the YBX1 in RCC. A 293 T cells were co-transfected with oe-AR and oe-lncTCFL5-2 or oe-YBX1 and oe-lncTCFL5-2, RIP detected the interaction between YBX1 and lncTCFL5-2. B 293 T cells were co-transfected with oe-YBX1 and four deletions mutants of lncTCFL5-2, RIP detected the interaction between YBX1 and the four mutants. C OSRC-2 cells were lentivirally transduced with sh-lncTCFL5-2 or oe-lncTCFL5-2, and then exposed to hypoxia or normoxia for 2 days. Western blot analysis of the expression of YBX1. D OSRC-2 cells were lentivirally transduced with pWPI or oe-lncTCFL5-2, then treated with 10 mg/ml cycloheximide (CHX). YBX1 protein levels were analyzed by western blot (lower right panel). The lower left panel is a graphic representation of YBX1 metabolic stability. MG132 (20 µM) was added in the OSRC-2 cells, Western blot analysis of the expression of YBX1. E SW839 and OSRC-2 cells were lentivirally transduced with sh-AR or oe-AR, respectively, and then cells were exposed to hypoxia or normoxia for 2 days. Western blot analysis of the expression of YBX1. F OSRC-2 cells were lentivirally transduced with oe-AR or oe-AR together with wildtype or mutant lncTCFL5-2. Sphere formation assays were performed to evaluate the CSC phenotype. After 14 days of incubation, colonies in five random fields per well were counted under a microscope. G OSRC-2 cells were lentivirally transduced with pLVTHM or wildtype or mutant lncTCFL5-2. Total RNAs were analyzed for CSC markers CD24, CD133, PAX2, CD105, and SOX2 by real-time PCR. H ACHN and OSRC-2 cells were lentivirally transduced with sh-lncTCFL5-2 or were co-transduced with sh-lncTCFL5-2 and oe-YBX1, and then cells exposed to hypoxia for 2 days. Sphere formation assay were performed to evaluate the CSC phenotype. After 14 days of incubation, colonies in five random fields per well were counted under a microscope. I SW839 cells were lentivirally transduced with sh-lncTCFL5-2 or co-transfected with sh-lncTCFL5-2 and oe-YBX1 and then cell exposed to hypoxia and normoxia for 2 days. Total RNAs were analyzed for CSC marker CD24, CD133, PAX2, CD105, SOX2 by real-time PCR (ab: Antibody, pLVTHM: Addgene plasmid 12,247; sh-lncTCFL5-2: The pLVTHM plasmid encoding lncTCFL5-2 shRNA, oe-lncTCFL5-2: The plasmid encoding lncTCFL5-2 sequence, oe-YBX1: The plasmid encoding YBX1 sequence; N: normoxia, H: hypoxia)