Fig. 3: MIF and its nuclease activity are required for DNA replication and DNA synthesis in cancer cells.

a Expression of endogenous MIF and exogenous Flag-tagged WT or E22A MIF proteins in parental and MIF knockout KO MDA-MB-231 cells. b Cell cycle distribution analysis by flow cytometry in parental, MIF-KO2, KO2-MIF, and KO2-E22A MDA-MB-231 cells after double thymidine (2 mM) synchronization followed by 1, 3, and 4 h fresh medium incubation. c Representative images of BrdU (red) and DAPI (blue) staining in parental, MIF-KO2, KO2-MIF, and KO2-E22A MDA-MB-231 cells after double thymidine (2 mM) synchronization followed by 2 h fresh medium incubation. Scale bar, 20 μm. d, e Quantification of BrdU-positive cells (d) and BrdU foci numbers per cell (e). n = 50 cells in each group were counted over three biologically independent experiments. Foci numbers >20 were counted as BrdU-positive cells. Statistical significance was determined by one-way ANOVA Dunnett’s multiple comparisons test. f Representative images of stretched DNA fibers in parental, MIF-KO2, KO2-MIF, and KO2-E22A MDA-MB-231 cells after IdU (25 μM, 20 min) and CIdU (250 μM, 20 min) incubation. Scale bar, 10 μm. g, h Distribution of the track lengths of CIdU and fork progression are indicated in different groups. n = 66 fibers in the parental group; n = 56 fibers in MIF-KO2 group; n = 42 fibers in the KO2-MIF group and n = 54 fibers in KO2-E22A group were analyzed from three independent experiments. Statistical significance was determined by one-way ANOVA Dunnett’s multiple comparisons test. i, j Mutation frequency analysis including deletion, insertion, and point mutations (i) and misincorporation rate analysis (j) in MIF-WT and MIF KO MDA-MB-231 cells using HPRT mutation assay (mean ± SEM, n = 3 biologically independent experiments). Statistical significance was determined by two-tailed Student’s t test. k, l Mutation frequency (k) and misincorporation rate (l) determined by HPRT mutation assay in parental and MIF KO DLD1 and DLD1-MSH6 rescued cells (mean ± SEM, n = 3 biologically independent experiments). Statistical significance was determined by one-way ANOVA Dunnett’s multiple comparisons test.