Figure 1.

SCUBE1 Is Enriched in Pulmonary ECs and Down-Regulated by Triggering Factors Driving PAH

(A) Abundance of secreted SCUBE1 protein in conditioned media from human pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs) was determined by immunoblotting (n = 3 samples per group). (B,C) PAECs were treated with control small, interfering ribonucleic acid (si-NC) or small, interfering ribonucleic acid specific to BMPR2 (si-BMPR2) for 72 h after which (B) SCUBE1 messenger ribonucleic acid (mRNA) expression was measured by reverse transcription, quantitative polymerase chain reaction of cellular homogenates (n = 3 samples per group) and SCUBE1 protein abundance was quantified by (C) enzyme-linked immunosorbent assay of conditioned media (n = 4 samples per group). (D,E) Similarly, PAECs were stimulated with hypoxia or interleukin (IL)-1β for 48 h, after which SCUBE1 (D) mRNA (n = 3 samples per group) and (E) secreted protein (n = 3 samples per group), were quantified by reverse transcription, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. (F) Immunoblot of PAEC homogenates (n = 3 samples per group) after the above-mentioned treatments. Immunoblots are representative of 3 independent experiments with band intensities quantified by densitometry. Values are presented as mean ± SD. The p values were calculated by Student's t-test, 1-way analysis of variance with post hoc Bonferroni test. The comparisons with p > 0.05 were not explicitly stated in the panels. AU = arbitrary units; PAH = pulmonary arterial hypertension.