Figure 2

Inhibition and activation of Notch signaling, respectively, in both MCF-7 and MDA-MB-231 cells. Normal group: MCF-7 or MDA-MB-231 cells were incubated under normal conditions; shNC group: MCF-7 or MDA-MB-231 cells were stably transfected with NC shRNA; shNotch1 group: MCF-7 or MDA-MB-231 cells were stably transfected with Notch1 shRNA; normal + Jagged1: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h; shNC + Jagged1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h after stable shNC transfection. Jagged1 + shNotch1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h and then Notch1 shRNA was transiently transfected for an additional 48 h. (A), (C), and (E) Total protein was isolated for western blot analysis using Notch1, Hey1, Hes1, and NF-κB65 antibodies. β-Actin was used as a loading control. (B), (D), and (F) Total RNA was extracted, and the expression levels of Notch1, Hey1, Hes1, and NF-κB65 were assayed by real-time PCR. The expression of every target gene was quantified using GAPDH as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).