Fig. 3.

ERK, JNK, and P38 MAPK signaling mediate the regulation of Notch4 on HPASMCs proliferation, apoptosis, and migration. A The MAPK signaling pathway were detected in HPASMCs transfected with siRNA against Notch4 or negative control siRNA. (n = 3–6) B Cell viability was examined in HPASMCs transfected with Notch4 plasmid or negative control plasmid together with ERK (U0126), JNK (SP600125), or P38 (SB203580) MAPK pathway inhibitors by using Cell Counting Kit-8 assay. (n = 4) C, D Proliferation was assessed by Edu assay in HPASMCs transfected with Notch4 plasmid or negative control plasmid together with U0126 (15 μM), SP600125 (15 μM), or SB203580 (15 μM) for 24 h. (n = 4) Magnification, × 100; Bar, 100 μm. E, F Migration was determined in HPASMCs transfected with Notch4 plasmid or negative control plasmid together with U0126, SP600125, or SB203580 for 24 h. Magnification, × 100; Bar, 50 μm. (n = 3) G, H The Annexin-V/PI assay was performed in HPASMCs transfected with Notch4 plasmid or negative control plasmid together with U0126, SP600125, or SB203580 for 24 h. Analyses of apoptosis including early apoptosis (Annexin-V positive and PI negative) and late apoptosis (Annexin-V positive and PI positive) were shown. (n = 4) Data were presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, comparison with normoxic HPASMCs treated with si-NC; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, comparison with hypoxia HPASMCs treated with si-NC. One-way ANOVA followed by Tukey’s correction for post-hoc comparisons were performed for A–H. CCK-8 cell counting kit-8, ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, MAPK mitogen-activated protein kinase, si-NC negative control short interfering RNAs (siRNA), si-Notch4 siRNA against Notch4, vector negative control plasmid, Notch4 Notch4 plasmid