FIGURE 1.

Real-time monitoring of Atf1 activity in living cells. A, live cell monitoring of Atf1 activity in wild-type (wt) and Δpyp1 cells in response to H₂O₂ and heat shock by using the firefly reporter assay. Wild-type cells and Δpyp1 cells transformed with the firefly reporter plasmid (3xCRE::luc(R2.2)) were incubated with d-luciferin and treated with H₂O₂ (1–4 mm) or incubated in 42 °C for 10 min before detection. Relative light units are expressed as the ratio of light emission of each sample to the basal (without stimulation) light emission of wild-type cells in EMM at 120 min. The data shown are representative of multiple experiments. B, live cell monitoring of Atf1 activity in wild-type, Δpyp1, and Δatf1 cells in response to ROS-generating agents by using the Renilla reporter assay. The cells as indicated were transformed with the 3xCRE::Renilla reporter plasmid, incubated with coelenterazine, and treated with CdCl₂ (2 mm), H₂O₂ (2 mm), heat shock (42 °C, 10 min), or LG (0.1% glucose), as described under “Experimental Procedures.” C, the Δpyp1 cells showed a higher basal reporter activity than wild-type cells. Values are from at least three independent experiments. Error bars indicate means ± S.D.