Figure 1: CRAF pS338 is induced by genotoxic stress and protects tumour cells from DNA damage.

(a) Embryonic fibroblasts isolated from wild-type (WT), BRAF^−/− or CRAF^−/− mice were irradiated (6 Gy), and DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci/cell±s.e.m. for n=6 fields analysed per group. ‘*’ indicates P<0.05 from two-sided t-test comparing WT and CRAF^−/−. Data shown are representative of two independent experiments. (b) HCT-116 cells were transfected with siRNA to BRAF and CRAF. Cells were irradiated (2 Gy) and cell survival was measured using clonogenic survival assay. Graph shows mean surviving fraction±s.e.m. ‘*’ indicates P<0.05 from two-sided t-test comparing si-CRAF to si-BRAF, si-CTRL or non-transfected with n=3 wells per group. Data shown are representative of three independent experiments. (c) HCT-116 cells treated with KG5 (1μm) overnight, irradiated (6 Gy) and whole cell lysates collected. Immunoblotting to indicate phospho CRAF and BRAF sites. Data shown are representative of three independent experiments. (d) Immunoblotting for pS338 CRAF following 6 Gy or 0.5 μM etoposide. Immunostaining for CRAF pS338 (green) with dose range of IR in HCT-116 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of five fields per group for two independent experiments. (e) HCT-116 xenograft tumours were irradiated (6 Gy) and then immunostained for CRAF pS338 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (f) Immunostaining for CRAF pS338 (green) in HCT-116 cells treated with KG5 (1 μM) overnight then irradiated (6 Gy). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of n=5 fields per group for two independent experiments. (g) HCT-116 cells were treated with KG5 and then irradiated. DNA double strand breaks were measured by neutral comet tail assay (n=100+ cells per group). Cell survival was measured using a clonogenic assay (n=3 wells per group). Bars represent mean±s.e.m. *P<0.05 from two-sided t-test comparing vehicle control and KG5. Data are representative of two independent experiments.