Figure 2.

Cdc7 depletion arrests IMR90 fibroblasts in G1 phase of the cell cycle. (a) Immunoblot analysis for Cdc7, its regulatory subunit ASK, and its substrate Mcm2 in control-siRNA (CO) and Cdc7-siRNA transfected (CDC7^KD) IMR90 whole cell extracts (actin loading control). (b) DNA content of CO and CDC7^KD cells was assessed by FACS analysis. Percentages of cells found to be in G1, S, and G2 phases of the cell cycle are indicated. (c) CO and CDC7^KD cells were stained for BrdU incorporation, and DNA was counterstained with propidium iodide (PI). Percentages of BrdU-incorporating cells are indicated. (d) Cell number was measured at the indicated time points (UT, untreated control). (e) Immunoblot analysis of Cdc7 protein levels and phosphorylation of Mcm2 at Ser-41 and at Ser-53 in cytoplasmic fraction (CF) and nuclear extract (NE) (actin and Orc4 loading controls). “Rescue” indicates IMR90 cells transfected with a CDC7 rescue plasmid. (f) Cells were measured for BrdU incorporation in CO, CDC7^KD transfected cells, as well as CDC7^KD cells treated with CDC7 rescue plasmid. Light blue indicates BrdU-incorporating cells, and dark blue indicates DAPI-stained nuclei.