S807 811

For Co-IP assay, cells were collected and lysed on ice with lysis buffer containing 0.5% NP40. The lysates were pre-cleared by incubation with protein A beads. The protein complex was then precipitated by a specific antibody together with protein A beads followed by extensive washing. The resulting materials were analyzed by western blotting as previously described^{81 (link)}. For IF analysis, PDLSCs were fixed using 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 4% BSA/PBS. Then the cells were treated with primary antibodies followed by incubation with FITC or TRITC–conjugated secondary antibodies. Coverslips were mounted in a glycerol/PBS solution containing DAPI. Confocal images were obtained using a Zeiss LSM confocal laser-scanning microscope. Antibodies against EZH2 (#5246), E2F1 (#3742), lamin A/C (#4777), H3K27me3 (#9733), H3K9me2 (#4685), H3K9me3 (#13969), RB and phospho-RB (S780, S795, and S807/811) (RB Antibody Sampler Kit #9969) were purchased from Cell Signaling Technology (CST). Antibodies against GAPDH (AC002) and α-Tubulin (AC007) were purchased from Abclonal. Antibody against CEMP-1 (ab134231) was from Abcam. Antibody against CAP (sc-53947) was from Santa Cruz. Antibodies used in IF were: EZH2 (612667, BD Pharmingen), H3K27ac (A7253, Abclonal), MED1 (A300-793A, Bethyl) and VAV2 (YT4864, Immunoway).

The cell count and viability were measured using the MTT assay. The cell cycle distribution was assessed by propidium iodide incorporation. Rb phosphorylation was assessed by western blotting (Cell Signaling; 4H1 and S807-811). The area under the curve (AUC) was estimated using Graphpad Prism v7.0 for palbociclib (0–1 μM), CX5461 (0–1 μM), and trametinib (0–25 nM). The responses to melphalan, bendamustine, FAS and TRAIL-R agonist antibodies, PRIMA-1^Met, dexamethasone, RITA, ABT-737, and ABT-199 were previously reported [6 (link), 9 (link)–14 (link)]. Results were scaled (mean-centered and standardized) to provide a z-score.

PD-0332991 was purchased from Selleckchem (Houston, TX, USA) and prepared as stock solutions in dimethyl sulfoxide (DMSO). Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). The antibody against CDK2 was purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA) and β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies were obtained from Jackson IR Laboratories, Inc., (West Grove, PA, USA). Protease inhibitor mixture, Triton X-100 and other chemicals were purchased from Sigma-Aldrich. The enhanced chemiluminescence detection kit was obtained from Amersham Biosciences (Piscataway, NJ, USA).