FIGURE 2.

Reduced inhibitors of DNA-binding proteins (ID)1/ID3 expression in congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) induced pluripotent stem cells (iPSCs) with bone morphogenetic protein type II receptor (BMPR2) mutations (mut) and the coordinative regulation between ID1 and ID3. a) Representative immunoblot analysis of the pSmad1/5, ID1 and ID3 levels in CHD-PAH mut and control iPSCs treated with or without 30 ng·mL⁻¹ BMP4 for 2 h. α-TUBULIN was used as loading control. b) Representative immunoblotting of BMPRII, pSmad1/5, ID1 and ID3 in the right ventricles of wild-type (WT) and Bmpr2^+/– rats. The results from three rats (marked as 1/2/3) are presented simultaneously. GAPDH was used as the loading control. c) Immunoblot analysis of human embryonic stem cells (hESCs) cultured with or without 30 ng·mL⁻¹ BMP4 for 2 h. α-TUBULIN was used as the loading control. d) Immunoblot time-course analysis of pSmad1/5, Samd1, ID1 and ID3 expression in hESCs stimulated without (0 h) and with 30 ng·mL⁻¹ BMP4 for 0.5–36 h. Smad1 and α-TUBULIN were used as loading controls. e) Immunoblot analysis of the ID1 and ID3 levels in hESCs stimulated with DMSO or LDN193189 (LDN) at different concentrations for 24 h. Smad1 and α-TUBULIN were used as loading controls. f) Immunoblot and densitometric analyses of total ID1 (endogenous (lower band) and exogenous (upper band)) and ID3 expression in the 3xFlag-ID1 inducible overexpression line h9-ESC-3F-ID1 treated with Dox (2 µg·mL⁻¹). α-TUBULIN was used as the loading control. g) Immunoblot and relative densitometric analyses of total ID3 (endogenous (lower band) and exogenous (upper band)) and ID1 expression in the 3xFlag-ID3 inducible overexpression line h9-ESC-3F-ID3. α-TUBULIN was used as the loading control. All cells were cultured in E8 medium (a and c–g). E8: Essential 8 Medium (composition shown in supplementary table S8); Dox: doxycycline.