Combination therapy with UCN-01 and gemcitabine induces DNA damage and apoptosis in TNBC cells. (A) Protein samples were collected 24 hours after drug treatment to detect changes in DNA damage (gamma-H2AX), checkpoint activation (phos-CHK1 andtTotal CHK1) and cell cycle progression (Cyclin A) by immunoblot analysis. (B) Cell cycle changes were assessed by BrdU-labeling and propidium iodide staining 24 hour after drug treatment. (C) Percentage of cells at 48 hours in early apoptosis (Annexin V +/7-AAD-) and late apoptosis (Annexin V+/7-AAD+) with representative data (D). MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), M6 cells (gemcitabine 4 nM; UCN-01 20 nM). P-value based upon change from vehicle treatment (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).