Fig. 4: MIF is translocated to the nucleus and locates at sites of DNA replication in the S phase.
From: MIF is a 3’ flap nuclease that facilitates DNA replication and promotes tumor growth
a G1/S synchronization workflow using double thymidine treatment. b, c Representative images of MIF nuclear translocation at different time points after double thymidine treatment. The percentage of cells with nuclear MIF was quantified and shown in c (mean ± SEM). n = 11 for 0 h and 1 h each group, n = 12 for 2 h group, n = 10 for 4 h group, and n = 12 for 8 h group were analyzed over three biological replicates. Scale bar, 20 μm. ****P < 0.0001 vs 0 h group, by one-way ANOVA Dunnett’s multiple comparisons test. d MIF is recruited to the DNA replication fork determined by isolation of proteins on nascent DNA (iPOND) analysis with or without Thymidine (Thy, 10 μM) chase for 1 h after EdU treatment. Representative blots from three independent experiments are shown. e, f Reciprocal co-IP of endogenous MIF and PCNA in MDA-MB-231 cells. Representative blots from three independent experiments are shown. g–i Immunostaining of MIF and EdU (g) or PCNA (h) in MDA-MB-231 cells at 2 h after double thymidine synchronization. Representative images from three independent experiments are shown. Scale bar, 20 μm. Colocalization of MIF-EdU (n = 36 from three biologically independent experiments) and MIF-PCNA (n = 35 from three biologically independent experiments) were quantified as Pearson’s correlation coefficient (mean ± SEM in i). Cells with Pearson’s correlation coefficient (r) >0.5 is considered as the positive correlation of MIF-EDU or MIF-PCNA colocalization, r > 0.7 is considered as a strong correlation, and r < 0.4 is considered as a weak or no correlation.
