Fig. 6

BMP10 signaling in HUVECs induces a delayed activation of the P38 MAPK pathway via GADD45β expression. A Cells were stimulated with 10 ng/mL BMP10 or not (NS). Cell extracts were subjected to WB analysis using antibodies against pP38-Thr180/Tyr182, P38, pHSP27-Ser78/82, HSP27, pEps15-Ser796, Eps15, pSMAD1/5-Ser463/465 and HSP90. Data are presented as fold change of each sample ± SEM relative to NS 5 min, n ≥ 3. B HUVECs were treated either with scrambled siRNA (siCTL) or siSMAD4 and then stimulated with BMP10 or not for 30 min. Cell extracts were analyzed by WB was performed as in panel A. C HUVECs were pre-treated either with vehicle or the transcription inhibitor actinomycin D (Act.D, 5 μM) for 30 min, then stimulated with BMP10 or not for another 30 min. Cell extracts were analyzed by WB as in panel A. Data are presented as mean folds ± SEM of n = 3. D SMAD1 binding sites and number of overlaps with footprints within GADD45β promoter extracted from transcription factor target gene database (TFBS). E HUVECs were stimulated with BMP10 or not (NS) and GADD45β mRNA expression was then assessed by RT-qPCR. The level of GADD45β mRNA expression was normalized to HPRT and represented as fold induction of each NS and BMP10 stimulated sample relative NS 0 min ± SEM of n = 2. (F) HUVECs were treated with either scrambled siRNA (siCTL) or siGADD45β and then stimulated with BMP10 or not (NS) for 30 min. Cell extracts were analyzed by WB as in panel A. Data are presented as mean folds ± SEM of n = 3. Statistical analysis for panel A was performed using Kruskal Wallis with Sidak’s post-test. Statistical analyses for panels B, C and F were performed using two-way ANOVA followed by Sidak's multiple comparisons posttest. For all panels: *,#P < 0.05; **, ##P < 0.01; ***, ###P < 0.001. *: BMP10-vs-NS; #: siCTL-vs-siSMAD4 or siGADD45β, or Vehicle-vs-Act.D