Figure 5

A combination of bromocriptine treatment and overexpression of Caveolin-1 enhances apoptosis of GH3 cells. (A) GH3 cells were transfected with caveolin-1 for 24 hours and then treated with or without 30 μM bromocriptine for another 12 hours. Non-transfected cells were treated for 12 hours with ethanol alone (vehicle) or bromocriptine. Cells expressing exogenous Caveolin-1 were immunostained with anti-Myc antibody and then detected with anti-mouse IgG Texas Red-conjugated secondary antibody. Three hundred Caveolin-1 expressing cells were counted in each experiment and apoptotic cells were determined by nuclear fragmentation after staining with Hoechst 33342 dye. Data were expressed as mean ± standard deviation from 3 independent experiments (angular transformed for analysis, back-transformed for presentation). (B) Phosphorylation of Tyr14 was enhanced when GH3 cells were exposed to bromocriptine. Cells were administered 30 μM bromocriptine as indicated, 24 hours after pcDNA4-caveolin-1 transfection. Cellular protein was extracted and separated on 12% SDS-PAGE then subjected to Western blotting. Anti-caveolin-1 or anti-phosphorylated caveolin-1 (Tyr¹⁴) specific antibodies were used to measure total and phosphorylated caveolin-1 respectively. Cellular protein extracts from NIH3T3 cells exposed to H₂O₂ were used as a positive control to detect phosphorylated caveolin-1. Dash (-) indicates vehicle treatment. The arrowhead points to endogenous caveolin-1 and the arrow marks recombinant caveolin-1. Abbreviation: Br, bromocriptine. *P < 0.05 versus experiment for caveolin-1 transient expression only or bromocriptine treatment only.