Expression of cof1-10 and cof1-13 alleles stabilises F-actin and triggers Ras hyperactivation and ROS production. F-actin was stained using Rhodamine–phalloidin in fixed wild-type, cof1-10 and cof1-13 cells grown to either log or diauxic shift in YPD. (A) The localisation of active Ras was assessed in vivo in wild-type and cof1-10 cells grown to either log or diauxic shift in YPD. (B) ROS levels were assessed in wild type (COF1), cof1-10, cof1-10Δras2, cof1-13 and cof1-10Δras2 grown for 24 hours in YPD to diauxic shift using H2DFC-DA. (C) Western blot was carried out to confirm the deletion of RAS2 in cof1-10 and cof1-13 strains. (D) Cell death was assessed in wild-type, cof1-10 and cof1-13 colonies by growing on YPD agar plates containing 10 μg/ml Phloxine B and visualised using a GFP filter set under a dissecting microscope. Because Phloxine B is actively pumped out of healthy cells, its accumulation marks regions within the colony that possess dead or dying cells. Scale bars: 10 μm.