Fig. 3

Extraction of CAFs / NFs and the promoting effect of CXCL12 on tumor. (A) Immunofluorescence of α-SMA expression in CAFs and NFs. Scale bar = 50 μm. (B) Western blotting showing the identification of CAFs and NFs. α-SMA and FAP were marker genes for CAFs. Vimentin was marker gene for mesenchymal cells. (C) Detection of CXCL12 secreted levels in culture medium (CM) of NFs, CAFs and bladder cancer cells after 48 h of culture using ELISA kit. (D) T24 cells and UMUC3 cells were treated with CAF or NF culture medium (CM) for 0–24 h, detected by western blotting with PDL1. (E) T24 cells and UMUC3 cells were treated with CXCL12 for 0–12 h, detected by western blotting with PDL1. (F) After the T24 cells had been treated with PBS, CXCL12 (50 ng/ml), AMD3100 (10 µg/ml), or AMD3100 + CXCL12 for 24, 48 and 72 h, the cell viability was assessed using MTT assay. (G) Wound-healing assay and (H) transwell invasion assay were performed following treatment with PBS, CXCL12 (50 ng/ml), AMD3100 (10 µg/ml), or AMD3100 + CXCL12 for 24 h in T24 cells. The statistical results were from three independent experiments. Values are means with SD. Scale bar = 300 μm. (I) The images of resected tumors in control and AMD3100 groups. (J) The tumor growth curve of each group. Data were presented as means ± SD (n = 6). (K) The tumor weight of each group. Data were presented as means ± SD (n = 6). (L) Left: PDL1 expression in IHC of control and AMD3100 groups. Right: Quantitative IHC analysis of PDL1 staining (n = 4)