Alpha amanitin

Cells were cultured at 37 °C in a 5% CO₂ environment. MCF-10A cells were cultured in mammary epithelial basal medium (MEBM) supplemented with 0.5 mg/ml Hydrocortisone solution, 20 ng/ml EGF, 100 ng/ml cholera toxin, 100 μg/ml recombinant human insulin and bovine pituitary extract (Lonza, Portsmouth, NH). The development of MCF-10A cells inducibly expressing non-specific shRNA or shRNA against BRG1 was described in previous work (Imbalzano et al., 2013 (link)). Knockdown of BRG1 was induced by adding doxycycline (Sigma-Aldrich, St. Louis, MO) at 10 μg/ml final concentration for 48 h.
Transient transfection of plasmids was performed using Lipofectamine 2000 or 3000 reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Development of EGFP-SRm160 and mRFP-SRm160 plasmids was descried in our previous work (Wagner et al., 2003 (link)).
The LINC complex was disrupted by transfecting cells with GFP-KASH4(Roux et al., 2009 (link)). F-actin was disrupted with 0.2 μg/ml Latrunculin A (LatA, Cayman Chemical, Ann Arbor, MI) for 30 min before imaging. RNA polymerase II (Pol II) was inhibited with 50μg/ml alpha-amanitin (Santa Cruz Biotechnology, Dallas, TX) for 4 h.