Journal: Pulmonary Circulation

Article Title: Activin‐A Regulates Bone Morphogenetic Protein Signaling in Pulmonary Endothelial Cells Without Affecting Bone Morphogenetic Protein Type‐II Receptor Expression

doi: 10.1002/pul2.70095

Figure Lengend Snippet: Effects of activin‐A treatment of BMPR‐II expression and BMP downstream targets in PMECs and PAECs in low serum and supplemented media conditions. (A–G) Human pulmonary microvascular endothelial cells (PMECs, n = 3) and human pulmonary artery endothelial cells (PAECs) were treated with activin‐A (20 ng/mL; ActA) for 1, 6, and 24 h in low serum (0.1% FBS) conditions, where indicated. RNA was isolated and SMAD7 (A), ID1 (B), and BMPR2 (C) mRNA expression was assessed by normalizing to three housekeeping (HK) genes— BACT , B2M , and HPRT . (D) In PMECs, protein lysates were immunoblotted for phospho‐Smad1/5, phospho‐Smad2, total Smad1, total Smad2, and reprobed for β‐actin as a loading control. (E) Densitometry of the ratio between pSmad1/5 and total Smad1, and densitometry of the ratio between pSmad2/3 and total Smad2, both normalized to β‐actin. (F) In PMECs and PAECs, proteins were lysed after 6‐h ActA treatment and subsequently immunoblotted for BMPR‐II and reprobed for α‐tubulin as a loading control. (G) Densitometry of the ratio between BMPR‐II and α‐tubulin. (H–N) PAECs were treated with ActA (20 ng/mL) for either 1, 6, or 24 h in supplemented endothelial cell growth media, where indicated. RNA was isolated and SMAD7 (H), ID1 (I), and BMPR2 (J) mRNA expression assessed by normalizing to three HK genes. (K) PAECs ( n = 5) were treated with ActA for 6 h in supplemented media. Protein lysates were immunoblotted for phospho‐Smad3, using an antibody which cross‐reacts with phospho‐Smad1. Protein lysates were also immunoblotted for total Smad1 and total Smad3 and reprobed for β‐actin. (L) Densitometry of the ratio between pSmad1 and total Smad1, pSmad3 and total Smad3, normalized to β‐actin. (M) PAECs ( n = 6) were treated with ActA for 6 h in supplemented media. Protein lysates were immunoblotted for BMPR‐II and reprobed for α‐tubulin. (N) Densitometry of the ratio between BMPR‐II and β‐actin. (O) PAECs ( n = 3) were treated with or without Act‐A (20 ng/mL) and/or BMP9 (0.3 ng/mL) for 6 h in 0.1% FBS. Protein lysates were immunoblotted for BMPR‐II and reprobed for α‐tubulin. (P) Densitometry of the ratio between BMPR‐II and α‐tubulin. Two‐way ANOVA (A, B, and E). One‐way ANOVA (H, I, J, L, and P). Wilcoxon matched pairs test (I). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Error bars represent mean ± SEM.

Article Snippet: PAECs or pulmonary microvascular endothelial cells (PMECs; Promocell or Lonza) were maintained in Endothelial Cell Growth Medium (ECGM)‐2 plus 2% fetal bovine serum (FBS) or ECGM‐MV2 plus 5% FBS, respectively, including supplement mix and antibiotic‐antimycotic (penicillin, streptomycin, and amphotericin B; Invitrogen).

Techniques: Expressing, Isolation, Control