Brd3 a302 368a

Tumor tissues were obtained from tumor-bearing mice treated with one dose of BETd-260 5 mg/kg or vehicle control. The following antibodies were used for IHC: BRD2 (A302-583A, 1:250), BRD3 (A302-368A, 1:250), BRD4 (A700-005, 1:100) from Bethyl Laboratories (Shanghai, China); cleaved PARP-1 (Asp214) (5625, 1:100) and Ki67 (8D5) (9449, 1:500) from Cell Signaling Technology (CST, Shanghai, China). IHC was performed following a standard protocol. Briefly, the 5-µm sections were de-paraffinized with xylene, rehydrated in graded concentrations of ethanol, and boiled in antigen retrieval buffer (Abcam, Shanghai, China) in a microwave oven for 5 min. Slides were incubated with specific primary antibodies at room temperature for 2 h. After incubation, the slides were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated antibody (Invitrogen, Shanghai, USA) at room temperature for 30 min, followed by incubation with ABC (avidin-biotin complex, Vectorlabs, Shanghai, China) for 30 min and visualization by the addition of 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Dako Diagnostics Co., Ltd., Shanghai). Sectioned tissues were counterstained with hematoxylin, dehydrated through a graded series of alcohol into xylene, and mounted under glass coverslips. Images of stained slides were captured using a standard light microscope.

Antibodies to the indicated proteins were purchased from the following vendors: BRD4 (ab128874), BRD2 (ab139690), PSMD14 (ab109123) and PSMD1 (ab140682; all Abcam); BRD3 (A302-368A; Bethyl); tubulin (926-42211; LICOR Biosciences); PSMD4 (3846), PSMD2 (25430), biotin (5597) and H2B (12364; Cell Signaling Technology) and PSMA1 (BML-PW8100-0100; ENZO Life Sciences). IRDye-800 anti-rabbit (926-32211) and IRDye-680RD anti-mouse (926-68072) secondary antibodies were purchased from LICOR Biosciences. Bortezomib (179324-69-7) was purchased from Calbiochem. Streptavidin magnetic beads (88816), NeutrAvidin agarose beads (29200), Lipofectamine RNAiMAX transfection reagent (13778075), RNA-to-C_t 1-Step kit (4392653) and TaqMan gene expression assays were purchased from Thermo Fisher. An RNeasy Plus mini RNA extraction kit (74134) was purchased from Qiagen.

FBP1 wildtype (wt) and FBP1 mutant (G260R) constructs were kindly provided by Dr. Haojie Huang from the Mayo Clinic [11 (link)]. The pCMV4a-Flag-c-Myc plasmid was purchased from Addgene. FBP1 nes mutants were generated by adding a nuclear export sequence (LALKLAGLDIGS) to the FBP1 c-terminal using the KOD-Plus- Mutagenesis Kit (Toyobo, Japan). Bacterial expression vectors for GST-TRIM28 recombinant proteins were generated using the pGEX-4 T-1 backbone vector. FBP1 antibody (ab109732)was purchased from Abcam (working dilution 1:1000); beta-Tubulin (2128S) was from Cell Signaling Technology - (working dilution 1:5000); TRIM28 (ab10483) was from Abcam (working dilution 1:3000); c-Myc (5605P) was from Santa Cruz Biotechnology (working dilution 1:1000); BRD2 (ab139690) was from Abcam (working dilution 1:1000); BRD3 (A302-368A) was from Bethyl Laboratories (working dilution 1:1000); BRD4 (ab128874) was from Abcam (working dilution 1:1000). JQ1, MG132, I CBP112, puromycin and cycloheximide (CHX), were purchased from Sigma-Aldrich (Shanghai, China); MK 2206, Trametinib, GSK126, Ku55933, SB203580 and Palbociclibwere from Selleckchem (Houston, USA). Gemcitabine was obtained from Eli Lilly and Company (Indianapolis, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, USA).