FIG. 3.

The poly(ADP-ribosyl)ation of CTCF is important for the growth suppression and proliferation functions. (A) The mutant of CTCF deficient in PARylation (CTCF Mut4) abrogates CTCF function as a negative regulator of progression into S phase. To assess the number of cells in S phase, the BrdU assay was performed with 293T, MDA425, and HeLa cells transfected with 2.0 μg of either pEGFP-C1 (EV) or a plasmid expressing EGFP-CTCF WT or EGFP-CTCF Mut4. Forty-eight hours posttransfection, cells were incubated with BrdU reagent for 2 h. The cells were fixed and stained with the anti-BrdU antibody as described in Materials and Methods. A secondary anti-mouse TRITC-conjugated antibody, together with DAPI, was used to visualize proliferating cells. A growth-suppressive effect on all cell lines transfected with the CTCF WT plasmid compared to those transfected with the EV was observed. In contrast, transfection of the cells with the CTCF Mut4 plasmid had no marked effect on cell proliferation. Each bar shows an average of results from three experiments performed in triplicate. Error bars indicate standard deviations. (B) Fluorescence images of BrdU assay results for cells transfected with pEGFP-C1 (EV) or the plasmid expressing EGFP-CTCF WT or EGFP-CTCF Mut4. Following the BrdU assay as described above, the cells were visualized using fluorescence microscopy. Arrows indicate cells which are both EGFP-CTCF positive and proliferating (BrdU positive). A reduced number of proliferating cells is observed following transfection with the EGFP-CTCF WT vector, indicating a growth suppression effect. This growth-suppressive effect is not observed in the cells transfected with the EGFP-CTCF Mut4 vector, which show proliferation similar to that of the control cells (those transfected with the EV).