FIG 6.

WDHD1 depletion reduces rereplication and decreases MCM3 chromatin loading and expression. (A) RPE1-E7 cells were treated with thymidine for 16 h, harvested at different times after release from thymidine, and analyzed with flow cytometry. (B) RPE1-E7 cells were transfected with siRNAs targeting WDHD1 and treated with thymidine for 16 h followed by bleomycin treatment for 48 h after release for 2 h from thymidine. Cells were analyzed with flow cytometry. Cells with >4C DNA content were quantified (right panel). (C) RPE1 cells were subjected to ChIP assay using antibody against MCM3 (left panel). RPE1-E7 cells were transfected with siRNA targeting WDHD1 and cells were subjected to ChIP assay (right panel). (D) RPE1-E7 cells were transfected with siRNAs targeting WDHD1, and real-time PCR analysis was performed. Another set of cells (right panel) was analyzed for steady-state levels of MCM3 by Western blotting. (E) RPE1-E7 cells were transfected with siRNAs targeting MCM3. Cells with >4C DNA content were quantified (right panel). Data from an experiment representative of 3 are shown. *, P < 0.05; **, P < 0.01.