Fig. 3.

AR of ccRCC cells influences angiogenesis and lymphoangiogenesis. a Outline of co-culture system. Endothelial cells (HUVEC and HDLMVEC) were co-cultured with ccRCC cells with indicated treatments (with/without overexpression/knockdown of AR) for 72 h, and then the endothelial cells were harvested. After harvest, the endothelial cells were maintained in mixed conditioned medium (CM from co-culture system) with fresh media at the ratio of 1:1. b, d Wound-healing assays of HUVEC and HDLMVEC cells co-cultured with A498 b and SW839 d with altered AR expression at indicated time points. Scale bar in b, d 10 μm. c, e Quantitation of endothelial cells migration in wound-healing assay after co-culture with indicated ccRCC cells as described in b, d. f, h Transwell invasion assay of HUVEC (upper) and HDLMVEC (lower) after co-culture with A498 cells overexpressing AR (f) and SW839 cells with AR knocked down (h). After 24 h, the invaded cells were stained and five random fields per well were analyzed. Scale bar in f, h, 5 μm. g, i Quantitation of the transwell invasion described in f, h. j A representative image of tube formation assays of HUVEC and HDLMVEC after co-culture with A498 overexpressing AR. The HUVEC cells during coculture with A498 cells were also blocked with VEGF-A neutralizing antibody (R&D Systems) at the concentration of 0.08 µg/ml. The endothelial cells were pre-stained with Calcein AM at the concentration of 2 µM and monitored with fluorescence microscope. l Tube formation assay of HUVEC and HDLMVEC after co-culture with SW839 cells with AR knockdown. The SW839 cells were infected with lentivirus for AR and sh-VEGF-C were used for co-culture with HDLMVEC cells for the rescue assay. Scale bar in j, l, 10 μm. k, m Quantitation of the tube number described in j, l. The functional assays were all replicated four times. *p < 0.05, was considered statistically significant by t-test for two groups and ANOVA for more than two groups