Journal: Nature Communications
Article Title: Androgen receptor increases hematogenous metastasis yet decreases lymphatic metastasis of renal cell carcinoma
Figure Lengend Snippet: 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
Article Snippet: HDLMVEC cells were purchased from Cell Applications, Inc. (San Diego, CA) and maintained with endothelial cell growth medium in attachment factor solution pre-coated flasks.
Techniques: Co-Culture Assay, Cell Culture, Over Expression, Expressing, Quantitation Assay, Migration, Wound Healing Assay, Transwell Invasion Assay, Staining, Concentration Assay, Fluorescence, Microscopy, Tube Formation Assay, Infection, Rescue Assay, Functional Assay