Fig 1: Characterisation of the HKe3-wtKRAS, HKe3-mtKRAS, HKe3 and HCT116 cell lines. a The upper band shows a KRAS activity assay, which detects binding of KRAS to the RAS-binding domain of RAF1 (RBD pulldown assay). These data show that mtKRAS cells have higher active KRAS compared to HKe3-wtKRAS cells. Two bands are detected as the HKe3-wtKRAS and mtKRAS cells express HA-tagged and untagged KRAS. The detection of HA-tagged KRAS is shown in the fifth lane as a positive control. The lower band shows the detection of KRASG13D (using a KRASG13D-specific antibody) in the HKe3-mtKRAS cells (HA-tagged). KRASG13D is also detected in the HCT116 and HKe3 cells. b Total cell lysate analysis shows that total KRAS expression was higher in HKe3-mtKRAS cells compared to HKe3-wtKRAS cells. Although total MAP2K1 (MEK1) expression was similar between the four cell lines, pMAP2K1 (S217/221) was higher in mtKRAS cells indicating stronger activation of the MAPK/ERK pathway. AKT1/2 and pAKT1(T308) levels were similar in the four cell lines. c–e The rate of proliferation (c), colony formation (d) and wound closure (e) was significantly higher in HKe3-mtKRAS cells compared to HKe3-wtKRAS cells. As expected HKe3-mtKRAS cells exhibited a phenotype similar to the HCT116 cells. Error bars represent the mean ± SD. Statistical significance was assessed using a Student’s t-test