For MTT assays, 2×10³ cells are seeded in triplicate in 96 wells in their regular culture medium (containing PLX4720 for PRT lines). Next day, cells are washed twice with PBS and then the medium is replenished containing the indicated RAF inhibitor. Medium is changed 48 hours later and after a further 48 hours, 10 μL of 5 mg/mL MTT reagent is added to wells, and incubated for three hours. Formazan crystals are then solubilized overnight with a 1:10 dilution of 0.1 M glycine (pH 10.5) in DMSO. Wells are then analyzed at 450 nM in a Multiskan^? Spectrum spectrophotometer. Results depicted are normalized to DMSO conditions and are a composite of three independent experiments. Error bars shown are representative of the standard error of mean (SEM).

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

COLO205 tumour cells are cultured in DMEM 10% FBS 1% penicillin/streptomycin supplemented with bovine insulin, at 37°C. Balb/C nude mice, female, 6-8 weeks old, weighing approximately 18-22 g, are inoculated subcutaneously at the right flank with COLO205 tumour cells (5×10⁶) in 0.1 mL of PBS mixed with matrigel (50:50) for tumour development. The treatment is started when mean tumour size reach approximately 100 mm³, with eight mice in each treatment group randomized to balance the average weight and tumour size. B9 cells are expanded in DMEM 10% FBS 1% penicillin/streptomycin. Upon trypsinization the cells are washed three times with 20 mL RPMI, and after the final centrifugation are re-suspended, counted, and adjusted by volume to a final concentration of 5×10⁷ cells per millilitre. B9 xenografts are started by injection of 5×10⁶ cells subcutaneously in 6- to 7-week-old female nude Balb/c mice. Compound dosing starts when the average size of tumours reach 50-70 mm³. Animals are equally distributed over treatment groups (n=10) to balance the average tumour size and body weight. Animals are dosed orally for days 1-14 twice daily and days 15-28 once daily with vehicle, vemurafenib 50 mg per kg, or PLX7904 50 mg per kg. 12-O-tetradecanoylphorbol-13-acetate (TPA) is put on the skin of all mice twice a week during weeks 3 and 4 at a dose of 2 μg in 200 μL acetone.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Zhang C, et al. RAF inhibitors that evade paradoxical MAPK pathway activation. Nature. 2015 Oct 22;526(7574):583-586.  [Content Brief]

  [2]. Basile KJ, et al. Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors. Pigment Cell Melanoma Res. 2014 May;27(3):479-84  [Content Brief]

  [3]. Le K, et al. Selective RAF inhibitor impairs ERK1/2 phosphorylation and growth in mutant NRAS, vemurafenib-resistant melanoma cells. Pigment Cell Melanoma Res. 2013 Jul;26(4):509-17  [Content Brief]