Anti integrin β1 antibody

To perform blocking studies, 5 different media conditions were used: (i) regular VSMC media, (ii) VSMC media with 50
μg/ml anti-integrin β1 antibody (Fisher), (iii) VSMC media with 50 μg/ml anti-N-cadherin antibody
(Sigma-Aldrich), (iv) VSMC media with 50 μg/ml of anti-integrin β1 antibody and 50 μg/ml of anti-N-cadherin
antibody (both antibodies), and (v) VSMC media with 50 μg/ml of non-immune IgG (Sigma-Aldrich). non-immune IgG was used as
a negative control as it was not expected to affect cellular interactions and mechanical properties. The antibody concentration of
50 μg/ml was chosen as it has been shown to be effective blocking concentration in other studies (Mendrick and Kelly, 1993 (link); Sun et al., 2005 (link); Yiin et al., 2009 (link)). Cells were exposed to different media conditions as soon as they were seeded on the
substrate and were maintained in culture for a period of 5 days after which AFM nanoindentation studies were performed to study
mechanical properties. This culture period was selected as VSMCs have been shown previously to stiffen in the first 3–5
days of culture (Hemmer et al., 2008 , Hemmer et al.,
2009). After this initial stiffening period, cell mechanical properties remain stable for 7–10 days (Hemmer et al. 2009 , Deitch et al. 2012 (link)).

Antibodies used were as follows: α-SMA (1A4, Sigma-Aldrich); cleaved caspase-3 (5A1E, Cell Signaling); MCL-1, BCL-X_L, BAK, BAK, BIM, BID, tubulin, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling); and BCL-2 (Bcl-2-100, Life Technologies). Secondary antibodies were obtained from Invitrogen [Alexa Fluor 488 goat anti-mouse immunoglobulin G2a (IgG2a) and Alexa Fluor 555 goat anti-rabbit IgG1]. F-actin and nuclei were stained with Alexa Fluor 546–phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen), respectively.
Reagents used included PF-562,271 (Selleckchem), S63845 (ApexBio), ABT-263 (Active Biochem), ABT-199 (Active Biochem), and Y-27632, RGD peptide, CCG-1423, and anti–integrin β₁ antibody (Sigma). siRNA duplexes targeting human BCL-2, human BCL-X_L, human MCL-1, human BIM, human YAP, and human TAZ were obtained from Dharmacon Inc. using ON-TARGETplus SMARTpool (Thermo Scientific).
Nonreplicative recombinant adenovirus expressing human α-SMA fused to red fluorescent protein (Ad-α-SMA-RFP) or shRNA against α-SMA and GFP (Ad-GFP-U6-α-SMA-shRNA) were generated, amplified, and purified by Vector Biolabs. Table S1 lists the primer and siRNA sequences used in this study.