Fig. 6. Nuclei and mitochondrial DNA are dispensable for contagious apoptosis. (A) Representative immunofluorescence of contagious apoptosis triggered by enucleated HeLa Env cells. HeLa Env cytoplasts were pretreated or not with 1 µM STS, washed, and co-cultured overnight with healthy HeLa CD4. HeLa Env cytoplasts and normal HeLa CD4 were pre-stained with CellTracker® Green and CellTracker® Red, respectively, and syncytia were stained with the chromatin-specific dye Hoechst 33342. (B) Quantitative analysis of the data obtained as in A. HeLa Env cytoplasts were pretreated or not with STS (1 µM) or ActD (5 µM) and co-cultured with healthy CD4 cells in the presence or absence of zVAD.fmk. The phenotypic characteristics (mean ± s.d., n=3; *P<0.05; #P<0.005) of syncytia with PACC, i.e. nuclear apoptosis (NA) or low mitochondrial transmembrane potential (_m) were determined by staining with Hoechst 33342 or DiOC₆(3), respectively. (C) Representative images of syncytia formed by fusion of normal (⁺)HeLa Env cells mtDNA-depleted (°). HeLa Env or CD4 were pre-stained with MitoTracker® Green (MTG), as indicated, and _m was assessed by TMRM staining. The yellow color indicates an overlap between the green (MTG) and red (TMRM) staining, observable in control syncytia, but not in contagious apoptosis in which the _m is lost. (D) Quantitative analysis of data obtained in C. Nuclear apoptosis and loss of _m were determined as previously. Values are means of two experiments ± s.d.