Acta2

The following primary antibodies were used: YAP (Cell Signaling, #14074, Cell Sugnaling Technology, Danvers, Massachusetts), phospho-YAP (Ser127) (Cell Signaling, #4911), LATS2 (Bethyl Laboratories, #A300-479A, Bethyl Laboratories, Montgomery, Texas), phospho-LATS2 (Thr1041) (Cell Signaling, #8654), atrial natriuretic peptide (Abcam, #ab180649, Abcam, Cambridge, United Kingdom), TEAD1 (Sigma-Aldrich, #AV39521), MYH7 (Sigma-Aldrich, # M8421), ACTA2 (Sigma-Aldrich, #A5228), OSM (Santa Cruz, #sc374039, Santa Cruz Biotechnology, Dallas, Texas), OSMR (Santa Cruz, #sc30011), cardiac troponin T antibody (Abcam, #ab33589), sarcomeric actinin (Abcam, #ab68167), CD45 (Abcam, #ab10558), CD68 (Abcam, #ab31630), Ly6G (Abcam, #ab25377), and α-tubulin (Sigma-Aldrich, #T6199). Immunoblot analyses and histological analyses were conducted as described previously (13) (link).

For cryosectioning, excised tissue was embedded and flash-frozen in O.C.T medium (Tissue Tech) using a dry-ice slurry/2-methylbutanol mixture and cryosectioned between 8-10um. Tissue cryosections were washed with PBS and fixed with 4% PFA for 10 min. Post fixation, slides were washed three times for 5 min each with PBS followed by permeabilization using 0.2% Triton-X100 in PBS (PBT) for 10 min. Slides were then blocked (5% BSA, 2% normal goat serum in PBT) at room temperature for 1hr. Post block, tissue sections were then incubated in primary antibody overnight at 4°C in blocking solution (3% BSA, 0.2% Triton-X100 in PBS). Primary antibodies used include pan-Laminin (Abcam, #ab11575), Acta2 (Sigma, #A2547), F4/80 (Invitrogen, #MF48000), Cdh1 (BD Bioscience, #610181), and ZO-1/Tjp1 (Abcam, #ab221547). After overnight incubation, slides were washed with PBS, and then incubated with goat ALEXA-Fluor 594- or ALEXA-Fluor 488- conjugated antibodies (Thermo Fisher Scientific) for 2 h at room temperature in blocking solution. Cell nuclei were counterstained with DAPI (Molecular Probes) and mounted in 4% (w/v) propyl gallate anti-fade solution. Immunofluorescent images were obtained using an SP5 confocal microscope (Leica). Acta2 and Cdh1 confocal image quantification analysis was performed as noted above (Okamura et al., 2014 (link); Pennathur et al., 2015 (link)).

Tissues were collected and fixed in paraformaldehyde 4%. Paraffin sections were treated with xylene, ethanol 100%, ethanol 90%, and ethanol 70% baths. Permeabilization was done in Triton 0.1%. Antigen retrieval was done in citrate buffer (ThermoFisher). Primary antibodies were Acta2 (Sigma-Aldrich), CD31 (R&D Systems), collagen-I (Abcam), eNOS (BD Biosciences), GFP (Abcam), laminin (Sigma-Aldrich), pSmad2 (Millipore), TGFβ (R&D Systems), VEGFR2 (Cell Signaling), and PV1 (Novus Biologicals). Pictures were taken using an SP5 confocal microscope (Leica).