The elevated expression of APP engaged in breast cancer cell proliferation. (A) APP expression is detected by 22C11 mouse monoclonal anti-APP antibody in human breast cancer cell lines and correlates with increasing malignancy. (+); a positive control of APP protein overexpressed in neuronal cells. (B) The expression of APP is compared in mouse breast cancer cells with increasing metastatic potential. (C) APP protein expression was present at a similar level in both M-IV and MDA-MB-231. Knock down of APP expression was verified in RT-PCR following lentiviral infection encoding shAPP in MDA-MB-231. APP knockdown resulted in decreased expression of APP and soluble APP. The equal volume of conditioned media was condensed by using Centricon and analyzed in Western blot. For the loading control, β-actin was uesd. (D) Cells (2x103) were seeded in 6-well plate and cell numbers counted using coulter counter at day 2 and 4. (E) MDA-MB-231 cells were seeded at two different numbers and the cell growth was compared by MTT assay. (F) MDA-MB-231 cells fixed and stained with propidium iodide (PI) were subjected to cell cycle analysis by FACS.