Figure 2: CRAF kinase activity is not required to drive radioresistance.

(a) HCT-116 cells were stably transfected to express wild-type CRAF (WT) or the phospho-mimetic CRAF S338D mutant. Cells treated with or without 6 Gy were analysed for DNA damage using clonogenic (*P=0.012, n=3 wells per group, two-sided t-test) and comet tail assays (P<0.0001, n=100+ cells per group, two-sided t-test). Bars represent mean±s.e.m. Data are representative of two independent experiments. (b) Immune-compromised nu/nu mice were implanted s.c. with tumour cells to each thigh, and only the right thigh received three fractions of 6 Gy on Days 5, 7 and 9. Graph shows mean tumour volume±s.e.m, *P=0.04 from two-sided t-test comparing WT+IR (n=10) versus S338D+IR (n=9) at the endpoint on Day 15. (c) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy. DNA damage was assessed by clonogenic (P=0.002, n=3 wells per group), comet tail (*P=0.0005, n=100+ cells per group) and γH2AX assays (*P=0.0007, n=6 fields per group). All bar graphs show mean±s.e.m. P values from two-sided t-tests comparing WT versus each CRAF mutant. Data are representative of two independent experiments. (d) CRAF^−/− MEFs were transfected with GFP-tagged WT, S338A, S338D or K375M CRAF for 72 h and then given 6 Gy. DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci per cell±s.e.m. for n=50+ cells analysed per group. *P<0.05 from two-sided t-test comparing WT and CRAF^−/−. Data are representative of three independent experiments.