Figure 2. Analysis of segregation of sister centromeres during meiosis I. Wild-type pat1⁺ (JG12226) cells carrying cen2-GFP (cen2-proximal loci were visualized by the LacI-GFP fusion protein bound to lac operator array inserted about 5 kb from cen2)⁸ were crossed to cells lacking cen2-GFP (JG15458) and sporulated in EMM2-NH₄Cl medium. Diploid pat1-as2/pat1-as2 (JG16022) and pat1-as2/pat1-as2 mat-Pc (JG16113) cells carrying heterozygous cen2-GFP were cultured to mid log phase in YES-Ade medium, then transferred to EMM2-NH₄Cl medium for 16 h at 25°C to synchronize cells in G₁, then shifted into EMM2 medium and released into meiosis by adding 25 μM 1-NM-PP1 inhibitor at 25°C or by shifting the cells to 34°C. Mating pheromone-signaling was triggered either by addition of synthetic P-factor (70 µg/ml) or expression of the mat-Pc as indicated. Cells in anaphase I or metaphase II were fixed, stained with Hoechst 33342 and antibodies against tubulin and GFP, and examined under a fluorescence microscope. Segregation of chromosome II labeled with cen2-GFP was scored in 600 anaphase I cells and in 200 metaphase II cells.