Figure 3

Effects of MIF knockdown on the S-phase of the cell cycle. The Click-iT™ EdU flow cytometry assay was used to measure the numbers of cells entering S-phase over a 3 h period. Dual parameter plots compare cellular DNA content (7AAD) against Edu incorporation with S-phase cells denoted by the inset box in each plot. Representative analyses for (A) MelCV and (B) Me1007 cells are presented after 3d of transfection with control (NC) or MIF siRNA duplexes. Quantitation of the percentage of cells in S phase in (C) MelCV and (D) Me1007 cell lines after MIF knockdown or treatment with control siRNA duplexes. (E) Analyses in (C) and (D) were repeated for an additional 4 melanoma cell lines. Values shown represent the proportion of cells entering S phase after MIF siRNA treatment normalised against NC siRNA treatment. MIF depletion significantly reduced the number of cells entering S-phase for 4/6 cell lines (means +/-S.E.M. from 5 independent experiments, ***p < 0.001 **p < 0.01 *p < 0.05 comparing between treatments using Student’s t-test).