Ro5126766

BGB‐283, compound C, and pimasertib were synthesized in‐house and exceeded a purity of 99% as measured by proton nuclear magnetic resonance (HNMR), liquid chromatography–mass spectrometry (LC‐MS), and high‐performance liquid chromatography (HPLC). Compounds were purchased from following source: vemurafenib, WuXi AppTec (Shanghai, China); selumetinib, PD‐0325901, and trametinib, BioChemPartner (Shanghai, China); and RO5126766, Active Biochem (Kowloon, Hong Kong). Stock solutions of compounds were prepared in dimethyl sulfoxide. Antibodies used were obtained commercially from the following sources: anti‐B‐RAF (SC‐5248), Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐C‐RAF (610152), BD Biosciences (San Jose, CA, USA); antibodies to MEK (9122), MEK1 (2352), phospho‐MEK1/2 (Ser217/221) (9154), ERK (4695), phospho‐ERK1/2 (Thr202/Tyr204) (4370), GAPDH (2118s), and anti‐rabbit IgG horseradish peroxidase (HRP)‐linked secondary antibody, Cell Signaling Technology (Danvers, MA, USA); and anti‐mouse IgG HRP‐linked secondary antibody (A0168), Sigma‐Aldrich (St. Louis, MO, USA). BALB/c nude mice (female) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All procedures involving animals were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of BeiGene.

The cell lines used were as follows: RWPE-1 (prostate epithelial cells); LNCaP, LAPC4, PC3, C4-2B, and DU145 (prostate cancer cells); 293T (human embryonic kidney cells); HeLa (human cervical adenocarcinoma cells, obtained from ATCC, Manassas, VA); PrEC (human primary prostate epithelial cells, obtained from Lonza, Basel, Switzerland); and ARCaP_E and ARCaP_M (Novicure Biotechnology, Birmingham, AL). All cell lines were tested and found to be free of mycoplasma and were cultured for no more than 10 passages according to the manufacturer’s recommendations. All cell types were checked for proper morphology prior to every experiment and consistently monitored for changes in cell replication that might suggest Mycoplasma contamination. Cell synchronization was performed as described previously (Yuan et al., 2014 (link)). The inhibitors used were as follows: RO5126766 (Active Biochem, Maplewood, NJ), nocodazole (Sigma-Aldrich, St. Louis, MO), BI2536 (Selleck Chemicals, Houston, TX), cycloheximide (Sigma-Aldrich), MG132 (Sigma-Aldrich), chloroquine (Sigma-Aldrich), recombinant human TGF-β1 (Thermo Fisher Scientific, Grand Island, NY), and recombinant human EGF (BD Biosciences).