A heterotrimeric SMARCB1–SMARCC2 subcomplex is required for the assembly and tumor suppression function of the BAF chromatin-remodeling complex

To co-express SMARCB1 and SMARCC2, DNA fragments corresponding to human SMARCB1 (full-length residues 1-385) and SMARCC2 (residues 1-958) were polymerase chain reaction (PCR)-amplified from the cDNA library of the Human kidney epithelial (HEK) 293T cell line. SMARCB1 and SMARCC2 were cloned into two multiple cloning sites of an in-house modified version of pETDuet-1 vector (Novagen) separately and sequentially. A Thioredoxin (Trx)-his6 tag and a PreScission Protease restriction site are present at the N-terminus of the first and second multiple cloning sites, respectively, in the in-house modified version of pETDuet-1 vector. The resulting proteins contained a Trx-his6 tag on the N-terminus of both SMARCB1 and SMARCC2.


Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) measurements were performed on a MicroCal TM Isothermal Titration Calorimeter iTC200 (GE Healthcare) in 50mMTris, pH8.0 and 200mM NaCl. For the protein of SMARCB1  , 88 μM of SMARCC2  was titrated into 6 μM of SMARCB1  . For the mutant protein of SMARCC2 (325-518) (R487C), 1.16 mM of SMARCC2  was titrated into 24.5 μM of SMARCB1  . For the protein of wild-type SMARCC1  , 102 μM of SMARCC1  was titrated into 12 μM of SMARCB1  . For the mutant protein of SMARCC1 (35A3-542) (R512Q), 155 μM of SMARCC1  was titrated into 9.5 μM of SMARCB1  . For the protein of Trx-Rpt1 and Trx-Rpt2, 300 μM of Trx-Rpt1 and 232 μM of Trx-Rpt2 were titrated into 46 μM of Trx-SMARCC2 SWIRM , respectively. The titration consisted of an initial injection of 0.4 l followed by 26 injections of 1.5 l every 120 s at 25℃. The titration data and binding plot after baseline subtraction were analyzed with the MicroCal Origin software with the two-site and one-site-binding models for SMARCB1  or SMARCC1  and SMARCC2   (R487C) or Trx-Rpt1, respectively.

NMR experiments
All NMR spectra of protein samples were recorded at 298 K on a Bruker AV600 NMR spectrometer equipped with a QCI cryoprobe. The protein samples were in 50mM Tris, pH8.0 and 200mM NaCl.

Co-immunoprecipitation
HEK293T cells were transfected with the indicated combinations of plasmids. 24 hours after transfection, HEK293T cells were lysed using ice-cold cell lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 8% glycerol, 0.5% NP40, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktails) and cleared by centrifugation at 13,000 rpm for 20 min at 4°C. The supernatants were then incubated with agarose conjugated anti-GFP antibody for 30 min at 4°C. The agarose beads were washed three times with cell lysis buffer and eluted with SDS sample buffer. Samples were then subjected to SDS-PAGE and western blot analysis.

Western blotting
The proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The membranes were subsequently blocked with 10% nonfat milk in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 1 h. For the samples of co-IP assays, the membranes were immunoblotted with anti-Myc (Sigma) and anti-GFP (Sigma) antibodies at room temperature for 1 h, and then probed with horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz) and developed with a chemiluminescent substrate (Millipore). Protein bands were visualized on the Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).

BrdU incorporation assay
Anti-BrdU staining was used to measure progression through G1 and entry in S phase.
The assays were performed by BrdU Labeling and Detection Kit I (Roche, USA) according to manufacturer's protocol. Briefly, Sorted BT-12 cells were plated at 40,000 cells per well on 8-well chambered slide and were cultured for 24 h. They were then incubated with complete medium supplemented with BrdU for 1 h. After fixation with 100% cold ethanol fixative, cells were stained with anti-Brdu antibody and anti-mouse Alexa Fluor® 555 secondary antibody, then slide were mounted and examined using fluorescence microscope. All experiments were performed in triplicates.

Plate colony formation
Sorted BT-12 cells were dissociated into single cells (1000 /well), were plated into 6-well plates and were allowed to grow for 14 days. The medium was changed every three days until the cells formed visible clone. Next, the media were removed, cell colonies were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet for 20 min, then cells were washed with water, air dried at room temperature for 1 h, and took a picture by Gel counter (Oxford Optronix, UK). All experiments were performed in triplicates.

Migration and invasion assays
Both migration and invasion assays were performed using a 24-well Transwell chamber system (Corning, USA). Sorted BT-12 cell were plated at 40,000 per well both for cell migration and cell invasion coated with matrigel (300 g/ml, 1 hr of solidification). Cells were incubated for 24 hr, fixed with 4% paraformaldehyde, stained by 0.1% crystal violet, and took a picture using an inverted microscope. All experiments were performed in triplicates.

Gene ontology enrichment and Gene set enrichment analysis
Gene ontology (GO) enrichment analysis was done by using Investigate Gene Sets (http://software.broadinstitute.org/gsea/msigdb/index.jsp) 10 . Selected terms from "hallmark gene sets" or "canonical pathways" were considered significant with p < 0.05.

Xenograft study
All of the procedures related to animal handling, care, and treatment in this study were performed following approval by the Institutional Animal Care and Use Committee of MD Anderson Cancer Center and following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. For the in vivo study, mice were subcutaneously inoculated at the left and right flank with BT-12 tumor cells (1×10 6 cells per injection) in 0.1 mL mixture of media and Matrigel (RPMI 1640/Matrigel, 1:1) for tumor development. Then we used the water containing doxycycline to raise the mice and changed the water containing dox every three days.