FIGURE 3.

Overexpression of porcine Bim_EL induces apoptosis of granulosa cells in vitro. A, cells plated on glass bottom 35-mm dishes were 80% confluent when transfected with pEGFP-N1-Bim_EL (panels a and b) or pEGFP-N1 (panel c, control) constructs. Note that Bim_EL-GFP was localized to the cytoplasm at 2 h post-transfection, and apoptotic phenotypes measured using Hoechst 33342 staining could be found in pEGFP-N1-BimEL-transfected cells at 8 h post-transfection (arrow). However, GFP-only expressing cells consistently had intact nuclei, and GFP was distributed throughout the cells. B, Western blot (WB) analysis of protein expression for endogenous and exogenous Bim_EL in granulosa cells transfected with a full-length Bim_EL-GFP fusion construct or mock vector. Transfected granulosa cells lysates were subjected to SDS-PAGE and immunoblotting using the anti-Bim antibody. Molecular masses of the protein bands are shown on the left side. Actin was used as fractionation control. * indicates an alternative splicing form of Bim_EL. C, caspase-3 activity was determined by caspase assay in GFP only or Bim_EL-GFP transfected granulosa cells (n = 4). Values represent means ± S.E. of three experiments. **, p < 0.01.