Spontaneously immortalized mouse embryo fibroblasts (MEFs), primary mouse VSMCs, Cas-mutant MEFs, and SYF-null MEFs were cultured as previously described (13 (link), 63 ). Cas-null and Cas-mutant MEFs were generously provided by Amy Bouton (University of Virginia) and Steven Hanks (Vanderbilt University). SYF-null (MEFs deficient in Src, Yes, and Fyn) and c-Src-reconstituted SYF-MEFs were purchased from ATCC. Primary mouse VSMCs were isolated by explant culture from 8–10 week old male C57BL/6J mice as described (64 (link)). To synchronize MEFs and VSMCs in G0, near-confluent cells were serum-starved by incubation in DMEM with 1 mg/ml heat-inactivated fatty-acid free BSA for 48 hours. The serum-starved cells were trypsinized, centrifuged, and resuspended in serum-free DMEM for 30 minutes at 37°C. Then, cells were replated on fibronectin-coated hydrogels with fresh growth medium (13 (link), 63 ) containing 10% FBS.
In some experiments, cells were plated in the presence of 0.03 µM jasplakinolide (Calbiochem) or 0.5 µM latrunculin B (Calbiochem) with 10% FBS. For FAK or Rac pharmacologic inhibitor experiments, near confluent MEFs in 35-mm culture dishes were serum-starved for ~48 hours and then stimulated with 10% FBS and either (vehicle), 20 µM FAK inhibitor (PF573228; Tocris Bioscience), or 150 µM Rac inhibitor (NSC23766; Santa Cruz) for selected times up to 20 hours. Although previous publications have used lower doses of these inhibitors (65 (link), 66 (link)), we used dose response curves for FAK autophosphorylation and Rac-GTP loading, respectively, in MEFs to select the concentrations of PF573228 and NSC23766 used in our experiments (Fig. S8A–B ). PF573228 was dissolved in DMSO as a 750× stock; vehicle- and PF573228-treated cells contained the same amounts of DMSO.
siRNA transfections for both MEFs and VSMCs were performed as described with Lipofectamine 2000 (63 ) in OPTI-MEM using final siRNA concentrations of 150–200 nM. After 4–5 hours of siRNA transfection, cells were allowed to recover overnight in fresh DMEM containing 10% FBS and then serum-starved in BSA-containing DMEM for 48 hours. All siRNA-based experiments were performed 72 hours after transfection. A non-specific Control Duplex X siRNA (target sequence: 5’NNATTCTATCACTAGCGTGAC-3’) was used as control (Dharmacon). Adenoviruses were titered and used as described (63 ). FRNK, FAK397F, and RacV12 were generous gifts from Christopher Chen (Boston University). Adenoviruses were used at the following multiplicities of infection: FRNK (600), FAK397F (1500), wild-type FAK (300), RacV12 (900), RacN17 (100) and Cyclin D1 (300). Adenoviruses encoding LacZ or GFP were used as controls.
In some experiments, cells were plated in the presence of 0.03 µM jasplakinolide (Calbiochem) or 0.5 µM latrunculin B (Calbiochem) with 10% FBS. For FAK or Rac pharmacologic inhibitor experiments, near confluent MEFs in 35-mm culture dishes were serum-starved for ~48 hours and then stimulated with 10% FBS and either (vehicle), 20 µM FAK inhibitor (PF573228; Tocris Bioscience), or 150 µM Rac inhibitor (NSC23766; Santa Cruz) for selected times up to 20 hours. Although previous publications have used lower doses of these inhibitors (65 (link), 66 (link)), we used dose response curves for FAK autophosphorylation and Rac-GTP loading, respectively, in MEFs to select the concentrations of PF573228 and NSC23766 used in our experiments (
siRNA transfections for both MEFs and VSMCs were performed as described with Lipofectamine 2000 (63 ) in OPTI-MEM using final siRNA concentrations of 150–200 nM. After 4–5 hours of siRNA transfection, cells were allowed to recover overnight in fresh DMEM containing 10% FBS and then serum-starved in BSA-containing DMEM for 48 hours. All siRNA-based experiments were performed 72 hours after transfection. A non-specific Control Duplex X siRNA (target sequence: 5’NNATTCTATCACTAGCGTGAC-3’) was used as control (Dharmacon). Adenoviruses were titered and used as described (63 ). FRNK, FAK397F, and RacV12 were generous gifts from Christopher Chen (Boston University). Adenoviruses were used at the following multiplicities of infection: FRNK (600), FAK397F (1500), wild-type FAK (300), RacV12 (900), RacN17 (100) and Cyclin D1 (300). Adenoviruses encoding LacZ or GFP were used as controls.
