(A) Scheme for removal of Pcr1 (REIII binding factor) in the ΔKHSS strain OFF isolate (ΔKHSS-OFF). Progeny of the cross was selected for ΔKHSS-OFFΔpcr1 genotype and identifiable colonies immediately grown for cytometry, and passaged for 456 hr. (B) Stable inheritance of repression in ΔKHSS-OFFΔpcr1. ΔKHSS-OFFΔpcr1 or ΔKHSS-OFF cells (dark blue lines) where analyzed by flow cytometry over consecutive days, the break indicating passaging without analysis. Δpcr1 had no significant effect on ΔREIIIHSS (light blue lines). (C) LEFT: scatter plots with partial point transparency of ΔKHSS-OFF or ΔKHSS-OFFΔpcr1 early and late in the time course. RIGHT: In the middle of the time course (asterisk in (B)), ΔKHSS-OFFΔpcr1 were struck for single colonies. The scatter plots for one of the isolates is shown. (D) Scheme for removal of functional Seb1 in ΔREIIIHSS strain. Selection and growth as in A., total passaging time 96 hr. (E) Weak inheritance of repression in ΔREIIIHSSseb1-1 (light blue lines). Analysis as above, total time course 96 hr. Removal of both Seb1 and RNAi pathways (ΔKHSS-OFFseb1-1Δpcr1) does not affect maintenance of silencing (dark blue lines). (F) Scatter plots of ΔREIIIHSS at 24 and 96 hr and through the entire time course for ΔREIIIHSSseb1-1. In these scatter plots, X and Y values of each cell are represented by purple dashes along the corresponding axis.