Fig. 5

BMP10 signaling in HUVECs drives the activation of the P38-MK2 axis. A HUVECs were stimulated with 10 ng/mL BMP10 or not (NS) for 30 min. Cell extracts were subjected to western blotting (WB) analysis using antibodies against phosphorylated (p) P38-Thr180/Tyr182, P38, pHSP27-Ser78/82, HSP27, pEps15-Ser796, Eps15, pSMAD1/5-Ser463/465 and HSP90 (loading control). Quantification of phosphorylation for P38, HSP27 and Eps15 reflects the normalized signal for the phosphorylated protein to total protein content, presented as mean fold change (BMP10-vs-NS) ± SEM of n = 5 independent experiments. **P < 0.01 using Kolmogorov–Smirnov test. B and C HUVECs were pretreated with selective ALK1/2/3/6 inhibitor LDN193189 (LDN, 5 µM) (Panel B), P38 inhibitor SB203580 (SB, 10 µM) or MK2 inhibitor PF3622044 (PF, 5 µM) (Panel C), or left untreated (vehicle) for 30 min. Subsequently, cells were stimulated with 10 ng/mL BMP10 or not (NS) for an additional 30 min. Cell lysates were then analyzed by WB using the indicated antibodies and quantification was performed as explained in panel A. Data shown represent mean folds ± SEM of at least 3 independent experiments. D HUVECs were pretreated with P38 inhibitor SB203580 (SB, 10 μM) or left untreated (vehicle) for 30 min. Cells were then stimulated with 10 ng/mL BMP10 or not (NS) for 4 h. Real-time quantitative polymerase chain reaction analysis was then performed to determine mRNA expression of ID1, SMAD6, SELE (E-Selectin), PTGS2 (COX2), and HAS2. Target gene expression was normalized to HPRT using 2-ΔΔCt method and presented as fold induction (BMP10-vs-vehicle) ± SEM of n = 4 independent experiments. Statistical analyses for panels B, C and D were performed using two-way ANOVA followed by Sidak's multiple comparisons post-test. For all panels: *,#P < 0.05; **,##P < 0.01; ***,###P < 0.001; ****,####P < 0.0001. *: BMP10-vs-NS; #: vehicle-vs-inhibitor (LDN, SB or PF)