Mouse collagen 4

Maxinunc microtiter plates (eBiosciences) were coated with 0.5 μg human collagen I (Corning), human collagen VI (Abcam), bovine fibronectin (Corning), mouse collagen IV (Corning) and mouse laminin (Corning) at 4°C for overnight, and blocked with 3% BSA for 1 h at 37 °C. Recombinant His-tagged BBA33 protein was serially diluted 1:2 starting from 4 μM down to 62.5 nM in PBS, 0.1% Tween-20, and added to coated wells in triplicate and incubated for 1 h at 37 °C. After five washes in PBS, 0.1% Tween-20, a 1:6000 dilution of monoclonal antibody directed against poly-His (His₆) was added to each well and incubated for 1 h at 37°C. Following five washes in PBS, 0.1% Tween-20, a 1:4000 dilution of HRP conjugated antimouse IgG was added to each well and incubated for 1 h at 37°C. The wells were then washed in PBS, 0.1% Tween-20, after which 3,3′,5,5′-tetramethylbenzidine was added as substrate. The enzymatic reaction was stopped after 3 min using 0.16 M sulfuric acid (Thermo scientific), and the absorbance at 450 nm was determined.

The cells were cultured on tissue culture dishes coated with 3% mouse collagen IV (Corning) for iPSC-SMPs and 0.1% gelatin for primary SMCs. Cells were seeded with an initial density of 2×105 cells per sample in the growth media (Medium231 with smooth muscle growth supplement, Fisher Scentific). To maintain their phenotype, PDGF-BB (10ng/mL) supplemented the growth medium for iPSC-SMPs. After 3–4 days when cells approximated 80% confluency, the media was switched to the differentiation media (Smooth Muscle Differentiation Media, Cell Applications), supplemented with 2.5 ng/ml transforming growth factor- β-1 (TGFβ-1) for 10 days. At the end of 10 days, the samples were lysed and processed for total RNA using the GeneJet RNA isolation kit (Fisher Scientific) according to the manufacturer’s instructions. The total RNA was then transcribed into cDNA (Fisher Scientific) according to our previous methods^{20 (link)}. Primers for qPCR consisted of ACTA2, TAGLN, CNN1, MYH11, COL1A, and GAPDH (Fisher Scientific). Relative fold change expression was normalized to GAPDH housekeeping gene using the ΔΔCT method, as described in our previous methods^{21 (link),22 (link)} and averaged out of n ≥3 samples per group.