Fig. 7

BMP10 inhibits the G1/S cell cycle transition in HUVECs. A HUVECs were synchronized at the G1 phase by serum starvation for 48 h. Subsequently, cells were then either replenished with 5% FBS or 0.5% FBS in the presence or absence (NS) of 10 ng/mL BMP10 added overnight. Cells were then treated with EdU for 1.5 h, trypsinized, and labeled for EdU (S phase) and propidium iodide (PI). Left panel: flow cytometry analysis assessing the distribution of different stages of the cell cycle (G1, S, G2/M). Right panel: Quantification of different cell cycle stages. Data are represented as mean % of each stage ± SEM of n = 4 and analyzed using two-way ANOVA with Benjamin Hochberg multiple comparisons post-test. Red asterisks represent statistical significance from G1-phase population comparisons between different conditions, while green asterisks represent that for S-phase population comparisons. * P < 0.05; *** P < 0.001. B Schematic representation of different key proteins implicated in the G1/S transition of the cell cycle. C HUVECs were stimulated or not with 10 ng/mL BMP10 for 2, 4, or 8 h. RT-qPCR analysis was then performed to determine mRNA expression of E2F2, CCND1 and CCNA1. Target gene expression was normalized to HPRT mRNA level using 2-ΔΔCt method and presented as relative expression (%) (BMP10-vs-vehicle) ± SEM at each time point (n = 3). *, #and $ represents statistical significance (BMP10-vs-NS) for CCND1, E2F2 and CCNA1, respectively. Statistical analysis was performed using Kruskal Wallis with Dunnett’s post-test^*, ^#, $P < 0.05; ^##P < 0.01; ^*** P < 0.001; ^****, ^####, ^$$$$ P < 0.0001. D HUVECs were synchronized and stimulated as explained in panel A. Cell extracts were subjected to WB analysis using antibodies against pRB1-Ser807/811, CyclinD1, and P27. Quantifications were performed using HSP90. Data are presented as mean folds ± SEM of n = 4. *P < 0.05 using Kolmogorov–Smirnov test