Smarcc1 baf155

10 million TC32 cells were incubated with trabectedin or DMSO for the indicated time, washed, cross-linked in 1% formaldehyde for 10 minutes and quenched with 0.2 M glycine. The cells were collected in cold PBS with 1× protease inhibitor (Sigma Aldrich), lysed in 20 mM Tri-HCl (pH 7.5), 85 mM KCl, and 0.5% NP-40 for 15 minutes on ice with dounce homogenizing. Chromatin was sheared with the E220 evolution focused sonicator (Covaris) for 10 minutes. 10 μg solubilized chromatin was immunoprecipitated with 1 μg mouse IgG (Abcam #18394), or H3K27me3 (Abcam #6002), 1 μg rabbit IgG (Cell Signaling #2729S) or 1 μg H3K9me3 (Abcam #8898), 2 μg rabbit IgG or 1 μg SMARCC1/BAF155 (Cell Signaling #11956S). Antibody-chromatin complexes were immunoprecipitated with Magna ChIP Protein A+G magnetic beads (EMD Millipore) and washed. DNA was eluted with 100mM NaHCO₃, 1% SDS, and 1× proteinase K for 2-hours at 65°C followed by 10-minute incubation at 95°C. ChIP DNA was purified with QiaQuick purification kit (Qiagen). Purified SMARCC1 ChIP DNA was analyzed with ChIP-qPCR, described below. Purified H3K27me3 and H3K9me3 ChIP DNA was submitted for 2×75bp sequencing and analyzed as described below.

Primary antibodies used for immunoblotting, co-immunofluorescence (co-IF), and immunohistochemistry: ARID1A/BAF250A (Cell Signaling Technologies, D2A8U), VIMENTIN (Cell Signaling Technologies, D21H3) for co-IF with E-CADHERIN and immunohistochemistry, E-CADHERIN (Cell Signaling Technologies, 4A2), Cytokeratin 19 (Santa Cruz Biotechnologies, A-3), KI-67 (MIB-1) (Dako), Cleaved CASPASE 3 (A175) (Cell Signaling Technologies, 5A1E), HSP90 alpha (Invitrogen, PA3–013), VIMENTIN (Cell Signaling Technologies, 5G3F10) for immunoblotting and co-IF with HSP90. ARID1B/BAF250B (Cell Signaling Technologies, E9J4T), PBRM1/BAF180 (Cell Signaling Technologies, D3F7O), BRM (Cell Signaling Technologies, D9E8B), BRG1 (Cell Signaling Technologies, A52), SMARCC1/BAF155 (Cell Signaling Technologies, D7F8S), SMARCC2/BAF170 (Cell Signaling Technologies, D8O9V), BAF60a (Santa Cruz Biotechnologies, 23), BAF53 (Santa Cruz Biotechnologies, C-7), SMARCB1/BAF47 (Cell Signaling Technologies, D8M1X). Chemical reagents: NVP-AUY-922 (LC Laboratories).