Fig. 3. Cell-cycle-dependent effects on apoptosis in GA1410 orc2-1 cells. (A) Survival (left) and ROS production (right) in GA1410 orc2-1 cells after synchronization in G1 or S phase for 3 hours at 23°C followed by a shift to 37°C for 3 hours. Viability measurements were normalized to viability of cells synchronized in G1 or S phase in the absence of the temperature shift to account for minor effects on viability of synchronization regimens. (B,C) FACS measurements of temperature-dependent effects on DNA replication in cdc6-1 (B) and GA1410 orc2-1 (C) cells synchronized in G1 with mating factor and subsequently released into S phase. Cells were synchronized in G1 phase for 2 hours at 23°C and maintained in G1 phase for an additional 5 hours at this temperature or at 37°C (`G1'), or were synchronized in G1 phase for 2 hours at 23°C and then maintained at this temperature or at 37°C for an additional 3 hours followed by release into S phase for 2 hours at the same temperature (`G1>S'). (D) Effects of orc2-1p depletion on DNA replication in GAL–orc2-1 cells that regulate orc2-1 expression from a galactose-inducible and glucose-repressible promoter. (E) GA1410 orc2-1 cells arrest in S phase after shifting to 37°C for 6 hours. DNA is stained with DAPI. (F) Effects of temperature on the cell cycle in exponentially proliferating GA1410 orc2-1 cells. (G-I) Effects of temperature on viability of cdc6-1 (G) and GA1410 orc2-1 (H) cells, and on ROS production in GA1410 orc2-1 cells (I) synchronized in G1 phase and then released (`G1>S') or not released (`G1') into S phase using the same regimen described for FACS analysis (B,C).