FIG. 2.

Depletion of BACH1 and BRCA1 results in delayed G₁/S progression and accumulation of cells in G₁. (A) Flow chart depicting the sequence of siRNA transfections and S-phase analysis. (B) Western blot analysis of whole-cell lysate of HeLa cells treated with control, BACH1, or BRCA1 siRNA. Equal amounts of extract were loaded and probed with antibodies against BACH1 and BRCA1. Actin was used as a loading control. (C, D, and E) Cells transfected with control, BACH1, and BRCA1 siRNAs were synchronized at G₁/S and released into S phase, and the cell cycle progression was monitored at the indicated time points after release by flow cytometric analysis of the total DNA content. The percentage distribution of cells in the G₁, S, and G₂/M phases of the cell cycle in control and BACH1- and BRCA1-depleted cells, as determined by PI staining, is shown. Western blot analysis results of BACH1 and BRCA1 in the corresponding cells at different time points after release are shown under each panel. PCNA is shown as a loading control.