Figure 6 .

The Rho1–Ycf1 bimolecular fluorescent complex formation after exposure to H₂O₂. (A) BiFC assays were performed in the YCF1–VN strain (HPY1710), carrying (a) pRS316–YFP^C–RHO1 or (b) YCp–YFP^C (pHP1730). Cells were grown in SC−Ura at 30° and incubated with 2 mM H₂O₂ for 2 hr before imaging. Images were captured, analyzed, and presented as in Figure 4B. Bar, 5 μm. (B) BiFC assays were performed in HPY1710, carrying pRS426–YFP^C–RHO1. Cells were grown in SC−Ura at 30° and incubated with 2 mM H₂O₂ for 4 hr (+H₂O₂) or mock treated (no oxidant) before imaging. Images were captured, analyzed, and presented as in Figure 4B. Bar, 5 μm. (C, left) Fluorescence intensity of individual cells of HPY1710 with pRS426–YFP^C–RHO1 was plotted and quantified using ImageJ software: pixel intensity in untreated cells, 9.57 ± 5.4 (in a.u.) and in cells treated with 2 mM H₂O₂ for 4 hr, 15.1 ± 9.9 (in a.u.) (P = 0.0002). (C, right) Fluorescence intensity of each punctum in HPY1710 with pRS426–YFP^C–RHO1 was analyzed similarly: pixel intensity in untreated cells, 0.64 ± 0.1 (in a.u.) and in cells treated with 2 mM H₂O₂ for 4 hr, 0.94 ± 0.5 (in a.u.) (P = 0.002). (D) Localization pattern of the Rho1–Ycf1 bimolecular fluorescent complex was analyzed as in Figure 5B from strains HPY1710 with pRS316–YFP^C–RHO1, YCp–YFP^C, or pRS426–YFP^C–RHO1 after treatment with H₂O₂ for 4 hr and HPY1710 with pRS426–YFP^C–RHO1 after mock treatment. Data are from three independent experiments (n = 300–400), and mean (%) ± SD are shown.