Fig. 2.

Proangiogenic SCs induce endothelial cell proliferation and tube formation. (A) (i) Higher expression of pERK in HUVECs in response to a cell-free–produced FGF treatment (+FGF CF) compared to a cell-free reaction without DNA (−FGF CF); purified FGF and untreated cells are displayed in Western blot images. (ii) Band area quantification of pERK/total ERK values normalized to untreated control (n = 3). Floating bars represent min to max and mean values; *P = 0.0453, **P ≤ 0.0054. (B) (i) HUVECs were treated with SCs prepared with/without inclusion of the TRX-bFGF DNA vector (+/−FGF SCs, respectively). Increased cell proliferation was detected in +FGF SC treatment after 48 h using (ii) viability assay and (iii) cell nuclei counting. All treatments were normalized to untreated control(n = between 11 and 20). **P ≤ 0.0055; ****P < 0.0001. (iv) Representative confocal images of HUVECs treated with the indicated treatments. Live-cell cytoplasms were stained with Calcein-AM (green), and cell nuclei were stained with Hoechst (blue). Scale bar, 50 μm. (C) (i) Illustration of a cell culture well cross-section in the tube formation assay performed with GFP-expressing HUVECs that were applied with/without SC treatments. (ii) Whole-well representative images of GFP-expressing HUVECs imaged 18 h after seeding (Top) and the corresponding AngioTool software analysis images (Bottom). Scale bar, 1 mm. (iii) Computerized image analysis presents a significantly higher average vessel length in +FGF SC treatment than −FGF SCs and untreated control. Values were normalized to the untreated control values. The gray dot (+FGF SCs) represents an outlier. (n = 8 or 9). **P = 0.0014; ****P < 0.0001. All results are presented as mean ± SD; n represents the number of independent samples in each group. One-way ANOVA with adjusted P value in multiple comparisons tests was used for statistical analysis. min to max, minimum to maximum; ns, not significant; Ex, Excitation; Em, Emission; Ec, Endothelial cell.