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Fig. 1. Loss of Orc2p function leads to the production of ROS. (A) Wild-type (W303) and orc2-1 (GA1410) cells stained with PI (red) and the ROS probe H2DCF-DA (green) before and after shifting to the nonpermissive temperature of 37°C for 6 hours. (B) FACS detection of ROS and PI staining of GA1410 orc2-1 cells before and after incubation at 37°C for 6 hours. Data are plotted on logarithmic scales. Signals from unstained cells correspond to autofluorescence. (C) Temperature- and time-dependent production of ROS (green) and PI (red) signals in GA1410 orc2-1 cells. Signals from autofluorescing cells were disregarded during quantitation of ROS and PI signals. (D) Suppression of ROS production in GA1410 orc2-1 cells by ectopic expression of ORC2. (E,F) Suppression of ROS in GA1410 orc2-1 cells transformed with a plasmid expressing the co-chaperone Mge1p, but not in cells transformed with an empty vector control. (G) Western blot showing that orc2-1p is destabilized in a proteasome-dependent fashion within 30 minutes of a shift to 37°C. Glucose (glu) represses transcription of orc2-1 driven by a galactose-regulated promoter in the GAL–orc2-1 cells used in this experiment. MG132 is an inhibitor of the proteasome and was used at a final concentration of 250 µM. Mcm2p was used as a loading control. (H) Effects of proteasome inhibition on lethality of high temperatures in GA1410 orc2-1 cells. (I) Effects of proteasome inhibition on production of ROS in GA1410 orc2-1 cells.
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