Fig. 1. Inactivation of iPLA₂ inhibits INS-1 cell proliferation. (A) BEL inhibits INS-1-cell proliferation in a concentration-dependent manner. Cells (10⁵) were treated with different BEL concentrations for up to 6 days and counted daily. Error bars represent the mean ± standard deviation (s.d.) of three experiments. (B) Proliferation of BEL-treated cells recovers upon BEL withdrawal. INS-1 cells were cultured in the presence of BEL (15 µM) for 2 days and then continuously cultured with (red) or without (green) BEL. Untreated cells were used as controls (blue). Error bars represent standard deviation from the mean of three experiments. (C) Transient expression of ARD-iPLA₂-GFP reduces INS-1 cell proliferation. INS-1 cells (2x10⁵) were transfected with ARD-iPLA₂-GFP (5 µg/100 mm plate), then counted daily, starting 24 hours after transfection. Mock-transfected cells (no DNA) were used as controls. The cell lysates were prepared and analyzed for expression of ARD-iPLA₂-GFP over 6 days, by western blotting with anti-GFP and anti-actin antibodies (top). Graph shows analysed data (bottom). Error bars represent the mean ± s.d. of three experiments. (D) Expression of Mut-iPLA₂-GFP inhibits the proliferation of INS-1 cells. iPLA₂-expressing INS-1 cells were transfected with Mut-iPLA₂-GFP (labelled Mut or Mut-iPLA₂ at 10 µg/100 mm plate) or mock transfected (no DNA) with FuGENE. Counting of cells began on the second day after transfection (bottom). After counting, cells lysates were prepared and analyzed by western blotting for GFP and actin (top). Error bars represent the mean ± s.d. of three experiments. ^*P<0.05. (E) Expressing siRNA against iPLA₂ decreases cell proliferation. Mock transfected INS-1 cells, and those transfected with psiRNA-iPLA₂ were counted on the fifth day after transfection (bottom), and the cell lysates were prepared for Western blot analysis for iPLA₂ (top). ^*P<0.05. A scrambled siRNA construct was transfected to serve as a negative control. Error bars represent the mean ± s.d. of three experiments.