Figure 1.

Growth characteristics of Klf4-transfected Klf4^−/−MEFs in culture. (A) Cell proliferation rates of Klf4^+/+ and Klf4^−/−MEFs, transfected or not with various plasmids. Cells were initially seeded at 10⁵ cells/plate in 60 mm plates. On day 0, cells were transfected in triplicates with pmKLF4-pEGFP, GFP control, or mock-transfected in parallel. Number of cells was counted (three plates per transfection) every day over a total of 3 days. The growth rate of the MEFs from each transfection was determined at each time point and the values represent the mean number of cells per well. N = 3 (*** = p < 0.001 for Klf4^+/+ and Klf4^−/− cells transfected with Klf4-GFP compared to mock- or GFP-transfected Klf4^+/+ and Klf4^−/− cells, respectively). (B) Quantification of apoptotic Klf4^+/+ and Klf4^−/−MEFs following GFP or Klf4-GFP overexpression. At 24 h post-transfection, immunostaining for both cleaved caspase 3 and GFP was done and number of cells that are positive for both were counted. N = 3 (Difference did not reach significance for Klf4^−/− cells transfected with Klf4-GFP compared to GFP-transfected Klf4^−/− cells, p = 0.059). (C) Western blot analysis of Klf4, p53, p21, cyclin E, γH2AX, and β-actin in protein extracts at 24 h post transfection of Klf4^+/+ and Klf4^−/− cells with GFP or Klf4-GFP.