Figure 4: TNFα alters NOTCH expression.

(a) Representative immunoblots of cleaved/transmembrane intracellular (NTM) regions for NOTCH2 and NOTCH3 in human control dPASMCs following transfection with DharmaFECT1 alone (DH1), siBMPR2, or non-targeting siRNA control (siCP) and treatment with TNFα (1 ng ml⁻¹) and/or BMP6 (10 ng ml⁻¹) for 1 h as indicated. Reprobed for α-tubulin to ensure equal loading. The data shown are representative of three experiments. (b) Representative immunoblots of NTM regions for NOTCH2 and NOTCH3 in human HPAH dPASMCs following transfection with DH1 alone, siACVR2A, or siCP and treated with TNFα (1 ng ml⁻¹) and/or BMP6 (10 ng ml⁻¹) for 1 h as indicated. Reprobed for α-tubulin to ensure equal loading. The data shown are representative of three experiments. (c,d) Notch2 (c) and Notch3 (d) mRNA expression in lungs isolated from 8-9 week old Bmpr2^+/+, SP-C/Tnf/Bmpr2^+/+, Bmpr2^+/- and SP-C/Tnf/Bmpr2^+/− (n=4 per group) transgenic mice. Expression was normalized to Actb. (e) Representative images of immunohistochemical staining for NOTCH2, NOTCH3 and αSMA in lung sections from control and HPAH subjects. Scale bars, 100 μm. (f,g) Proliferation of human HPAH dPASMCs on day 6 following transfection with DH1 alone, siNOTCH2 (f), siNOTCH3 (g) or siCP and treatment every 48 h with TNFα (1 ng ml⁻¹) and/or BMP6 (50 ng ml⁻¹) as indicated (n=3 cell lines). (h,i) Proliferation of human control dPASMCs on day 6 following transfection with DH1 alone, siNOTCH2 (h), siNOTCH3 (i) or siCP and treatment every 48 h with TNFα (1 ng ml⁻¹) and/or BMP6 (50 ng ml⁻¹) as indicated (n=3 cell lines). One-way analysis of variance with post hoc Tukey’s for multiple comparisons used in c,d,f and i. *P≤0.05, **/^##P≤0.01, ***/^###P≤0.001. Error bars represent mean±s.e.m.