Figure 4.

Cdc7 depletion causes apoptosis in BT549 (triple-receptor negative) breast cancer cells. A: Time course of CDC7 mRNA knockdown (KD) in BT549 cells relative to cells transfected with control-siRNA (CO) (upper panel). Immunoblot analysis of untreated (UT), control-siRNA-, and CDC7-siRNA–transfected BT549 whole cell extracts (WCE) probed with the indicated antibodies (lower panel). B: At the indicated time points, cell number was measured in UT, CO, and Cdc7-depleted (Cdc7^KD) cell populations. The graph shows fold-increase in cell numbers calculated for each time point relative to the number of cells seeded. C: DNA content of Cdc7^KD cells at the indicated times (left panel). At 72 hours posttransfection, BrdU incorporation was assayed. DNA was stained with propidium iodide (PI). Merged images of the BrdU-FITC (green) and PI (red) channels show the percentage of BrdU-positive cells (center panel). WCE from UT, CO, and Cdc7^KD cells immunoblotted with the indicated antibodies (right panel). D: Cell death and fragmented apoptotic nuclei (inset) were analyzed by phase-contrast microscopy and positive TUNEL staining in Cdc7^KD and CO cells (left panel). DAPI was used to stain DNA. Apoptotic cell death was confirmed in Cdc7^KD cells by WCE immunoblotting with the indicated antibodies (right panel).