Figure 1.

bcl-2 counteracts apoptosis induced by gas3/PMP22. (A) bcl-2, crmA, gas2, and hPLAP were coexpressed with h-TR in NIH3T3 cells. Six hours after microinjection apoptosis was induced by serum deprivation. Sixteen hours later cells were fixed and processed for immunofluorescence to detect h-TR. Survival was scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (p < 0.001). (B) bcl-2, crmA, gas2, and hPLAP were coexpressed with gas3/PMP22 in NIH3T3 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22. Survival was scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (p < 0.001). (C) gas3/PMP22 and bax killer activity. Different amounts of pGDSV7S containing gas3/PMP22, bax, or gas2 were comicroinjected with pGDSV7h-TR (25 ng/μl) in the nuclei of NIH3T3 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect h-TR. Survival was scored as described in the text. Datarepresent arithmetic means ± SD for three independent experiments. (D) gas3/PMP22-dependent morphological changes: confocal generated overlay showing cellular and nuclear phenotypes in NIH3T3 cells coexpressing Gas3/PMP22 and Bcl-2 (A) or h-Tr and Bcl-2 (B) or P₀ (C). NIH3T3 cells 24 h after seeding were microinjected with pGDSV3-hTR (100 ng/μl) and pGDSV7-bcl-2 (50 ng/μl), with pGDSV7-gas3/PMP22 (100 ng/μl) and pGDSV7-bcl-2 (50 ng/μl), or with pEXV-P₀ (100 ng/μl). After 24 h cells were fixed and processed for immunofluorescence analysis to visualize Gas3/PMP22 (dA), h-TR (dB), or P₀ (dC) (green) using the specific antibodies. Propidium iodide was used to visualize nuclei (red). Images were overlayered using a Zeiss confocal microscope and are displayed in pseudocolor. Bar, 5 μm.