EWS/WT1-KTS and EWS/WT1 + KTS expression co-operates with loss or inactivation of p53 to transform MEFs. (A) Western blots of lysates from SV40 transformed MEFs expressing either eGFP, EWS/WT1-KTS or EWS/WT1 + KTS under the control of a 4-OHT inducible promoter 48 hours after 4-OHT treatment (upper panel) or a doxycycline repressible promoter without doxycycline (lower panel). The anti-WT1 antibody detects a Cterminal-epitope in WT1. Arrow indicates EWS/WT1 to distinguish it from endogenous WT1. A non-specific band of similar size to EWS/WT1 was observed in SV40-transformed, but not untransformed MEFs. (B) qPCR analysis of mRNA expression of eGFP, EWS/WT1-KTS or EWS/WT1 + KTS in MEFs induced by either the 4-OHT inducible or tetracycline repressible expression systems. (C) Fold change in cell number 14 days after MEFs transformed by either SV40 or EIA/RAS were infected with eGFP, EWS/WT1-KTS or EWS/WT1 + KTS using the 4OHT inducible system in SV40 transformed cells, and the doxycycline repressible system in EIA/RAS transformed cells. Cells were plated at equal densities and counted and re-plated at the same dilution every 3–4 days. Data represent the mean ± SEM of three independently generated pools of MEFs tested in three independent experiments. (D) MEFs derived from littermate wildtype (upper panel), p53+/- (middle panel) and p53-/-(lower panel) mice were infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS and then plated at equal density. Cells were counted and re-plated on the indicated days. Values are mean ± SEM of three independently generated and infected pools of MEFs tested over three independent experiments. # denotes a p value of 0.003 and * denotes a p value of 0.004 with Student’s t-test comparing eGFP to EWS/WT1–KTS and eGFP to EWS/WT1 + KTS. Representative images of morphology of MEFs at 22 days are shown.