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. Author manuscript; available in PMC: 2015 Jan 1.
Published in final edited form as: Angiogenesis. 2013 Oct 10;17(1):261–274. doi: 10.1007/s10456-013-9395-0

Fig. 2.

Fig. 2

SEMA4D-induced production of PDGF-B from endothelial cells causes differentiation, proliferation and migration of pericytes and their association with HUVECs. (a) C3H/10T1/2 were grown in media conditioned by HUVECs with and without serum, treated with sSEMA4D with or without anti-PDGF-B blocking antibody, and differentiation determined by immunoblot for RGS5, NG2, and αSMA. GAPDH was used as a loading control. (b) Immunoblot for RGS5, NG2, and αSMA in C3H/10T1/2 cells grown in media conditioned by sSEMA4D treated HUVECs, either controls (C) or HUVEC with Plexin-B1 silenced by shRNA (PB1shRNA). (c) Proliferation of C3H/10T1/2 cells determined by optical density of stained cells at 450nm (Y-axis) at 0, 2 and 4 days in media conditioned by HUVECs with and without serum treated with sSEMA4D, with silenced Plexin-B1 (Plexin-B1 shRNA), control IgG or anti-PDGF-B blocking antibody (α-PDGF-B). (d) Migration of C3H/10T1/2 cells towards 0.1% BSA (negative control) or media conditioned by untreated HUVECs (untreated) or HUVECs treated with sSEMA4D (sS4D) with or without silenced Plexin-B1 (PB1 shRNA), control IgG or anti-PDGF-B blocking antibody (α-PDGF-B). (e) Quantification of stained migration assay membrane, determined by pixel intensity (Y-axis). Error bars represent the standard deviation from three migrations per condition. (f) HUVECs (green) were co-cultured with hPC-PL cells (pericytes, red) in the presence of sSEMA4D (left three columns) or in media conditioned by HN12 cells (middle three columns) or HN13 cells (right three columns), either in control conditions (top row), or in the presence of anti-PDGF-B antibody (second row), where Plexin-B1 was silenced in HUVECs with shRNA (Plexin-B1 shRNA, third row) or incubated with C3 toxin (fourth row). Co-association of these cells is shown (merge, yellow). (g) Quantification of co-association assay (% counted HUVECs in 10 hpf associated with pericytes, Y-axis; *, p ≤ 0.05; **, p ≤ 0.01 for all graphs). (h) Immunoblot for Plexin-B1 (upper panel) in 293T transfected with a Plexin-B1 construct, hPC-PL cells and HUVECs, to rule out direct effects of SEMA4D on pericytes. GAPDH was used a loading control for all blots (lower panels).