Figure 4.

Notch signaling is required for proliferation and survival in breast cancer cell lines and regulates cyclin A, cyclin B1, and NOXA. A, left, Western blots showing that RNAi silencing of Notch-1 in MDA-MB231 cells also down-regulated Notch-4, whereas nearly quantitative silencing of Notch-4 by RNAi had no effects on Notch-1. Control was a siRNA with no homology with known mammalian genes. Right, RNAi silencing of Notch-1 (which also affects Notch-4) or silencing of Notch-4 alone inhibited the proliferation of MDA-MB231 cells. Data are representative of three experiments each conducted in triplicate. SDs where not visible were smaller than data point symbols. B, silencing of either Notch-1 or Notch-4 in T47D:A18 cells inhibits proliferation and potentiates the effects of 4-OH-TAM. T47D:A18 cells were transfected with siRNA to Notch-1 or Notch-4 or scrambled control siRNA. Twenty-four hours after transfection, cells were seeded in a 96-well plate (8,000 per well) and treated with decreasing concentrations of 4-OH-TAM (25–0.38 μmol/L) in 0.5% ethanol for 24 h. At the end of treatment, cytotoxicity was evaluated by crystal violet staining. C, RNase protection assay showing that mRNA levels for cyclins A and B1 were reduced in MDA-MB231 cells transfected with Notch-1 siRNA. L32 (rRNA) and glyceraldehyde-3-phosphate dehydrogenase were internal controls. Additional controls were untreated cells and tRNA (nonspecific protection control). D, Western blots showing that cells transfected with Notch-1 siRNA had dramatically reduced levels of cyclin B1 and cyclin A proteins, as well as increased NOXA expression, compared with controls. Left, MDA-MB231 cells (48 h after transfection); right, T47D:A18 cells (72 h after transfection).