Fig. 3. Formation of cytoplasmic SGs occurs independently from apoptosis. (A,B) SGs form before the _m is lost and phoshatidylserine residues are exposed on the cell surface. Cells treated to obtain maximal SG formation (1-3 hours HAsO₄, Figs 1, 2) do not manifest a loss of the _m, as detectable with the _m-sensitive dye DiOC₍₆₎3 (A), nor do they stain with Annexin V (B). (C) SGs form before cytochrome c (Cyt c) is released from mitochondria. After the indicated treatment, HeLa cells were stained for the simultaneous detection of Cyt c (green) and TIA-1 (red). Note that an 18 hour incubation period with HAsO₄ is required to detect diffuse or absent Cyt c staining in a fraction of cells. (D,E) Effect of the broad spectrum caspase inhibitor Z-VAD.fmk on SG formation. Cells were treated for the indicated period with HAsO₄, in the presence or absence of Z-VAD.fmk and the frequency (±s.d., n=3), of SG+ cells (D) or cells exhibiting nuclear apoptosis (E) was determined by staining with anti-TIA-1 or Hoechst 33342, respectively.