Figure 3.

Mitogen‐activated protein kinase 4 (MAPK4) silencing inhibits the proliferation of endothelial cells (ECs) by regulating the Raf/MEK/ERK1/2 signaling pathway. Human umbilical vein ECs (HUVECs) were transiently transfected with MAPK4 small interfering RNA (siRNA) (50 nM) in 24‐well plates via Lipofectamine 3000 reagent in vitro. (a) Volcano plot showing genes with differential expression in MAPK4‐silenced HUVECs (MAPK4 HUVECs) compared with NC HUVECs, as determined by RNA sequencing (RNA‐seq). n =  3 per group. (b, c) Gene set enrichment analysis plots (left) and heat maps (right) of the RNA‐seq data for NC HUVECs and MAPK4 HUVECs. (d) Western blot analysis was used to evaluate the levels of protein kinase B (AKT), phosphorylated AKT (p‐AKT), c‐Jun n‐terminal kinase (JNK), phosphorylated JNK (p‐JNK), nuclear factor κB (NF‐κB), phosphorylated NF‐κB (p‐NF‐κB), (e) ERK1/2, phosphorylated ERK1/2 (p‐ERK1/2), (f) rat sarcoma (Ras), Raf, p‐Raf, MEK, and phosphorylated MEK (p‐MEK) in HUVECs. (g) 24 h after transfection, transfected cells were treated with the p‐ERK1/2 inhibitor and cultured for another 24 h. (h) Immunofluorescence was used to evaluate and quantitatively analyze the p‐ERK1/2 level in HUVECs. Representative data from three independent experiments are shown. **p < 0.01. ERK1/2, extracellular regulated protein kinases 1/2; MEK, mitogen‐activated extracellular signal‐regulated kinase; Raf, rapidly accelerated fibrosarcoma.