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Fig. 6 CTGF participates in the R1881-induced VEGF accumulation in the cytosol, subsequently inhibiting cell migration. (a) Double-exposed immunofluorescence micrographs showcasing TGN 38 (a Golgi apparatus marker) in green and VEGF in red. In DMSO-treated control cells, VEGF-positive immunoreactivity is observed to co-localize with TGN 38-positive areas. Nuclei are highlighted by DAPI staining in blue. Scale bar = 20 μm. (b) Both R1881 (5 nM) and DHT (2 nM) increased the levels of VEGF and CTGF protein in HUVECs. Values shown in parentheses represent the quantitative results after adjusted with their own α-tubulin protein levels and expressed as fold of their own control. (n = 3). *p ＜ 0.05 different from corresponding control. (c) R1881 (0.05–10 nM) concentration-dependently increased the formation of VEGF-CTGF complex (top panel) and decreased the formation of VEGF-VEGFR complex in HUVECs (bottom panel). (d) Treatment with VEGF (100 ng/mL) or CTGF (10 μM) alone for 8 h enhanced the endothelial cell migration, and these effects disappeared when the cells were co-treated with VEGF and CTGF together. Values present the means of fold of corresponding control ± s.e.mean. (n = 3). *p ＜ 0.05 different from corresponding control. #p ＜ 0.05 different from the VEGF-treated group. §p ＜ 0.05 different from the CTGF-treated group. (e) Treatment with R1881 for 8 h reduced the endothelial cell migration, and this effect was abolished by pre-treatment with WP631, a CTGF inhibitor. Values present the means of fold of corresponding control ± s.e.mean. (n = 3). *p ＜ 0.05 different from corresponding control. #p ＜ 0.05 different from the R1881-treated group. CTGF, connective tissue growth factor; IB, immunoblotting; IP, immunoprecipitation; VEGF, vascular endothelial growth factor. (f) RhoA prevents the CTGF-reduced VEGF secretion in HUVECs. Transfection with RhoA V14 pcDNA increased the cleavage of CTGF and the complex formation of VEGF-CTGF (cleaved form), but reduced the complex formation of VEGF-CTGF (full length) in HUVECs. (g) Pre-treatment with WP631, a CTGF inhibitor, abolished the R1881-reduced the VEGF concentrations in the conditioned medium of HUVECs culture. Values present the means of fold of corresponding control ± s.e.mean. (n = 4). *p ＜ 0.05 different from corresponding control. #p ＜ 0.05 different from the R1881-treated group. Conc. concentration; IB, immunoblot; IP, immunoprecipitation;.