Fig. 4.

Effects of leukaemia inhibitory factor (LIF) on the proliferation of L6 cells in serum-free medium are mediated by increased c-Myc protein expression. (a) Representative western blot from three separate experiments showing c-Myc protein expression in L6 cells after different times of culture (12, 24 and 48 h) Dulbecco’s modified Eagle’s medium (DMEM; D), DMEM + LIF 1 ng/mL (L) and DMEM + 10% fetal bovine seum (FBS; F). (b) Immunofluorescence staining showing c-Myc protein expression in serum-starved L6 cells after 12 h culture in the presence of DMEM, DMEM + LIF 1 ng/mL and DMEM + 10% FBS. Immunostaining of c-Myc protein was performed in three separate experiments. Images were merged with 4′,6′-diamidino-2-phenylindole (DAPI) to show nuclear localization. Arrows indicate the nuclear localization of c-Myc protein. Bar, 100 μm. (c) Quantitative analysis of nuclear c-Myc-positive cells (cells costained with c-Myc and DAPI) after 12 h LIF treatment, determined in 10 random images for each treatment for each experiment. Graphs were plotted from data obtained from three separate experiments and show the mean ± SEM of values expressed as a percentage of values in the control (DMEM) group. **P < 0.01 compared with DMEM (one-way ANOVA and Tukey’s post hoc test).