Transfections Transfection of GH3 and A431 cells was performed using Lipofectamine Plus reagent according to the manufacturers protocol. Briefly, the day before transfection, 6 105 cells were plated on a 6 well cell culture grade Petri dish. One g DNA and 6 l Plus reagent were diluted into 100 l serum free medium and 4 l lipofectamine Inhibitors,Modulators,Libraries was added to 100 l serum free medium. Inhibitors,Modulators,Libraries these two pre comple es were then mi ed and incubated for 15 min at room temperature. The DNA Plus lipofectamine reagent comple was added to each well containing Brefeldin_A GH3 or A431 cells in fresh serum free medium. Cells were incubated at 37 C in 5% CO2 in air for 3 hours, then the old medium was replaced with fresh complete medium after incubation. The times after trans fection for immunocytochemical staining or TUNEL analyses are indicated in the results.

Immunocytochemistry For the analysis Inhibitors,Modulators,Libraries of Myc tagged caveolin 1 e pression in GH3 cells, cells were briefly washed with PBS and fi ed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were permeabilized by incubating with PBS containing 0. 5% Triton 100 for 10 min. The perme abilized cells were immersed in blocking solution con taining 10% normal goat serum in PBS for 1 hour. The cells were then incubated over night at 4 C with either anti caveolin 1 or Myc primary antibody. After three washes with PBS, cells were incubated with the secondary antibody for 2 hours at room temperature. Slides were mounted with Mowiol 4 88 and visualized by confocal laser scanning microscopy before being digitally photo graphed.

TUNEL assay The TUNEL assays were conducted as previously described with some modifications. Briefly, DNase I treated GH3 cells or cells ectopically e pressing caveolin 1 were washed twice with PBS and fi ed with 4% paraformalde hyde in PBS, pH7. 4, for Inhibitors,Modulators,Libraries 10 min at room temperature. Cells were permeabilized with 0. 1% Triton 100 in 0. 1% sodium citrate for 2 min on ice. Cell ectopically e pressing caveolin 1 were labeled with monoclonal anti Myc anti body, then visualized by Te as Red conjugated anti mouse IgG antibody. Cells were TUNEL labeled using the In situ Cell Death Detection Kit according to the manufacturers instruction. Caspase inhibitor treatment and quantification of cell apoptosis Treatment with caspase inhibitors and quantification of cell apoptosis were conducted as follows GH3 cells were seeded in a 24 well dish one day before transfection.

Cells were transfected with pcDNA4 caveolin 1 or pDsRed N1 by Lipofectamine Plus reagent. Transfected cells were treated with caspase inhibitors at 50 mM final concentra tion for 48 hours, then immunocytochemical and TUNEL assays were used to quantify apoptotic cells. Anti c Myc monoclonal antibody was used as the first antibody to recognize caveolin 1 e pressing cells, followed by Te as Red conjugated anti mouse IgG. Cells e pressing DsRed N1 were directly detected by fluorescent microscopy.