Figure 2. Suppression of NGAL expression causes increased proliferation and decreased apoptosis in IMCD cells.

mIMCD-3 cells were stably transfected with two shRNAs directed against NGAL (shRNA 1+2) or the scrambled control shRNA. (a) A total of 1×10⁵ cells were plated and cultured for 48 h in the presence of 10% FBS before trypsinization and counting; n=6 separate experiments for each cell type, *P<0.04 vs cells expressing the scrambled shRNA. (b) A total of 1×10⁴ cells per well were plated and cultured for 48 h in the presence of 10% FBS before incubation with the WST-1 reagent and detection of viable cells via determination of OD 415/655 (proliferation index); n=4 independent experiments, *P*lt;0.01 vs cells expressing the scrambled shRNA. (c) Cells at equal density were cultured in the presence of BrdU for 30 min before FACS analysis for BrdU incorporation; n=3 independent experiments, *P<0.03 vs cells expressing the scrambled shRNA. (d) Cells at equal density were serum starved for 48 h before labeling with Annexin V PE and quantification by FACS analysis; n=4 independent experiments, *P<0.05 vs cells expressing the scrambled shRNA.