Figure 8.

E4orf4-induced membrane blebbing and apoptosis is modulated by Src kinase activity. (A) 293T cells were transfected with 0.25 μg of Flag-GFP and 3 μg of empty vector (GFP+EV) alone, or together with 1 μg of activated mouse or chicken c-src (mcsrcY527F/ccsrcY527F) or 1 μg of mouse or chicken kinase-deficient c-src (mcsrcK295M/ccsrcK295R). In parallel experiments, similar transfections were performed except that the empty vector was replaced by myc–E4orf4 (2μg; GFP+E4orf4). 24 h after transfection, the cellular morphology of transfected cells was examined in live cells and typical blebbing phenotypes of E4orf4-expressing cells are presented on the right. Arrows show the “popcorn-like” morphology of GFP-positive cells cotransfected with E4orf4 and activated c-src plasmids that presented a higher number and size of blebs in individual cells, as compared with GFP-positive cells transfected with E4orf4+GFP only. At least 300 GFP-positive cells were counted for each transfection and the percentage of blebbing cells was determined from the number of GFP-positive cells that arbored more than one bleb per cell relative to the total number of GFP-positive cells (graphical representation on the right). The data are the means ± SE of at least three independent experiments. (B) The same cells were kept at 37°C for an additional 24 h (48 h after transfection), following witch non-adherent and adherent cells were washed in PBS and the nuclear morphology was analyzed by DAPI staining after cell fixation. Specimens were analyzed by fluorescence microscopy and representative GFP-positive cells (GFP staining) with the corresponding nuclear staining (DAPI) are presented for each transfection. Arrows show GFP-positive cells that presented typical E4orf4-dependent apoptotic nuclear morphology. Percentages of apoptotic nuclei were obtained by counting at least 300 GFP-positive cells for each condition and are expressed as the number of cells presenting nuclear condensation relative to the total number of GFP-positive cells. The data are representative of at least three independent experiments (means ± SE). (C) Before cell fixation, aliquots of each cell population were kept and lysed in SDS sample buffer. Expression levels of exogenous proteins were analyzed by immunoblotting with the corresponding antibodies as indicated and tyrosine phosphorylation of cellular proteins was visualized using RC20-HRPO antiphosphotyrosine for immunoblot analysis of equal amounts of total proteins run on SDS-PAGE followed by electrotransfer onto nitrocellulose (Ponceau staining of gels are shown as loading controls).