Figure 4.

Depleting the PP2A R2/B56 regulatory subunit reduces Drosophila S2 cell number and activates apoptosis. (A–C) Drosophila S2 cells were incubated in the absence (Control) or presence of dsRNA targeted to EGFP or to the specific phosphatase subunits indicated at the bottom of A–C. After 72 h, cells were photographed with phase-contrast optics. (Magnification ×32.) (A) Depleting the PP2A-A or -C subunit reduces cell number and increases membrane blebbing. Representative photographs are shown for cells treated with dsRNAs targeting EGFP or the PP2A A or C subunits. (Bar = 10 μm.) (B) Depleting the PP2A-A , -C subunit, or both R2/B56 regulatory subunits reduces Drosophila S2 cell number. dsRNA-treated cells from ×32 magnification fields were counted, and values represent the mean ± SE from four experiments treated with two different batches of dsRNA where at least three fields from each treatment were scored. (C) Depleting the PP2A-A, -C subunit, or both R2/B56 regulatory subunits induces membrane blebbing in Drosophila S2 cells. dsRNA-treated cells from magnification fields ×32 were scored for total cells and apoptotic (membrane blebbing) cells, and % apoptotic values represent the mean ± SE from three experiments treated with two different batches of dsRNA where at least three fields from each treatment were scored. (D) Depleting the PP2A-A, -C subunit, or both R2/B56 regulatory subunits stimulates caspase-3-like activity. Drosophila S2 cells were incubated in the absence or presence of 500 μM cycloheximide (CX) for 3 h, or dsRNA targeted to EGFP or to the specific phosphatase subunits indicated at the bottom for 3 days. Caspase assays were performed as described under Materials and Methods. Values represent the mean caspase-3 activity ± SE from four experiments. Caspase activity was normalized for protein concentration and expressed as a percent of EGFP dsRNA-treated cells.