APP involved in the induction of cell cycle inhibitor p27
in breast cancer cells. (A) Knock-down of APP in MDA-MB-231 cells using two different shRNA constructs of APP (shAPP-5 and shAPP-7) resulted in marked suppression of both cellular and soluble form of APP expression. The p27kip1 expression was elevated in shAPP-5 and shAPP-7 cells. (B) The p27kip1 and p21cip1 expression was evaluated in M-I and M-IV after introduction of shluc, shAPP-5, or shAPP-7. (C) The control and shAPP-7 cells were incubated in serum deprived medium for 3 hours and then released with 10% serum for the indicated time points. The cells were harvested and subjected to assessment of p27kip1 and p21cip1 expression. (D) The cells incubated in serum-free medium for 18 hours were treated with 10% serum for 60 minutes and then the images were acquired to show subcellular localization of p27kip1. The nuclear localized p27kip1 was confirmed by merging with DAPI images. The longer image acquisition was needed to detect p27kip1 in the control (shluc) cells due to the low expression of p27kip1. Scale bar = 20 μm.