Figure 3.

MM cell-derived Jagged stimulates bone marrow stromal cell (BMSC) angiogenic potential: (A) Activation of Notch signaling and VEGF-A expression induced in HS5 cells co-cultured with HMCLs^SCR or HMCLs^KD assessed by qRT-PCR of the relative gene expression variation for the Notch target gene HES1 and VEGF (normalized to HRPT) calculated by the 2^−ΔΔCt formula (data are expressed as the mean value ± SD). Statistical analyses were carried out by one-way ANOVA and Tukey post-hoc test; * is for p ≤ 0.05; ** is for p ≤ 0.01; *** is for p ≤ 0.001. (B) ELISA for VEGF-A secreted by co-culture systems of HS5 cells and HMCLs^SCR or HMCLs^KD. Data represent the amount of VEGF released by each culture normalized on VEGF expressed by HMCL cultured alone. In each sample, the amount of VEGF (pg/mL) was normalized to the cell concentration. (C) Tube formation assay of the HPAECs stimulated with CM secreted by co-culture systems of the HS5 cells and HMCLs^SCR or HMCLs^KD. 4X magnification images are shown. Graphs show the quantification of the number of areas, branch points and total tube length. (D) Adhesion to fibronectin of the HPAECs stained with Calcein-AM and treated with CM secreted by the co-culture systems of the HS5 cells and HMCLs^SCR or HMCLs^KD. The graph reports the intensity of the adherent fluorescent cells. (E) Migration of the HPAECs treated with CM of co-culture systems of HS5 cells and HMCLs^SCR or HMCLs^KD was assessed by wound healing assays. Representative pictures at 4X magnification. The graph shows the average open area of the wounds expressed in pixels. Statistical analyses were carried out by ANOVA and Tukey post-hoc tests; * is for p ≤ 0.05; ** is for p ≤ 0.01; *** is for p ≤ 0.001.