Fig. 2: AR enhances ccRCC VM formation via altering TWIST1 expression.

a The qRT-PCR assays for the 16 genes related to VM formation in 786O cells (left) transfected with AR-cDNA (oeAR) versus control (pWPI) and SW839 cells (right) transfected with shRNA-AR (shAR) versus mock (pLKO). b Western blot assay for AR and TWIST1 protein levels in SW839 cells (left) with pLKO or shAR and in 786O cells (right) with pWPI or oeAR. c–d Western blot assays were performed on SW839 cells (c) transfected as indicated as well as 786O cells transfected as indicated (d). e 2D Matrigel-coated VM assays were performed in 786O cells transfected as indicated (upper panels) and SW839 cells transfected as indicated (lower panels). f 3D collagen I-based VM assays were performed in 786O cells transfected as indicated (upper panels) and SW839 cells transfected as indicated (lower panels). g Western blot assays to detect TWIST1 protein levels in SW839 (left) and 786O (right) cells after treating with 10 μM or 40 μM Harmine for 48 h. h–i 2D Matrigel-coated (upper panels) and 3D collagen I-based (lower panels) VM assays were performed after treating 786O cells transfected with pWPI or oeAR with TWIST1 inhibitor, Harmine. j Representative H&E and IHC staining of TWIST1 in adjacent noncancerous tissues (left) compared to paired ccRCC tissues (right). k The IHC score of TWIST1 between 23 paired adjacent tissue and ccRCC tumors. l The mRNA level of TWIST in ccRCC samples (n = 534) and adjacent normal tissues (n = 72) from TCGA database. m The correlation between AR and TWIST1 level based on IHC score (n = 51). n Overall survival probability of ccRCC patients was negatively correlated with TWIST1 mRNA expression based on TCGA database. For (e) and (f), quantitations are at the right, and data are expressed as mean ± S.D. *p < 0.05 and **p < 0.01 compared to controls.