Fig. 2: Axl inhibition suppresses the proliferation and induces the cytotoxicity and apoptosis of human pulmonary arterial endothelial cells (hPAECs).

a HPAECs from healthy individuals exposed to 10 ng/ml of epidermal growth factor (EGF) with or without R428 at indicated concentrations. Control cells (co) were exposed to dimethyl sulfoxide (DMSO). Data from n = 3 biological independent experiments performed in triplicate are presented. Open, green, and dark gray circles define untreated control (co), EGF, and EGF + R428 conditions, respectively. b, c Cytotoxicity in donor and IPAH hPAECs was determined by detecting lactate dehydrogenase (LDH) release into the cell culture media [A_492nm–A_620nm]. Cells treated with Staurosporine (SSP) were used as a positive control. Data are reported as percentage release of LDH compared to DMSO-treated control (co) cells. Data from n = 3 biological independent experiments performed in triplicates are presented as mean ± SEM. Number of d Hoechst and propidium iodide (PI) e positive hPAECs after R428 treatment. Data from n = 4 (Hoechst) and n = 5 (PI) independent experiments presented as the n-fold change normalized to dimethyl sulfoxide (DMSO)-treated control cells (co). f, g Relative mRNA expression of f intercellular adhesion molecule 1 (ICAM1) and g vascular cell adhesion molecule 1 (VCAM1) normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) as a reference gene after R428 treatment. Data from n = 3-5 biological independent experiments are presented as the n-fold change (2^−∆∆Ct) compared with DMSO-treated control (co). P values for distinct conditions are given for their comparison with DMSO-treated control cells (co). Open and gray circles define DMSO- and R428-treated conditions, respectively. h Analysis of AXL, ICAM1, and VCAM1 after small interfering RNA (siRNA) knock-down of AXL in hPAECs. AXL, ICAM1, and VCAM1 mRNA expression were normalized to HPRT as a reference gene. Data from n = 3 biological independent experiments in triplicate are presented as the n-fold change (2^−∆∆Ct) compared with siRNA scrambled control (si co). Opened and black circles define si co and siAXL conditions, respectively. For statistical analysis in a–g one-way ANOVA with Tukey’s post hoc test for multiple comparisons ^*P < 0.05, ^***P < 0.001, and ^****P < 0.0001 versus DMSO-treated control cells; ^####P < 0.0001 versus EGF-treated cells were implemented; for h Student’s t-test ^*P < 0.05 and ^***P < 0.001 versus scrambled siRNA control-transfected cells. All the data are presented as mean ± SEM.