Figure 5.

The effect of iron on GSH-depleted cells. (A) YPD-grown gsh1Δ cells inoculated in SD medium lacking GSH, grown for the indicated number of divisions, and processed for Aco1 and Leu1 enzyme activities. Data are the mean of three independent cultures±s.d. (B) Same as in (A) with gsh1Δ cells carrying Myc–Yap1, and processed for redox western, using an anti-Myc antibody. (C) YPD-grown gsh1Δ cells inoculated in SD medium lacking GSH and grown for six divisions, and left untreated (black bar), or further incubated with GSH (1 mM) (dark grey bars) or FeCl₃ (100 μM) (light grey bars) for the indicated time (min). FET3 expression monitored by RT–PCR as in Figure 3A. Values are the mean of triplicate samples of the same experiment±s.d. (D) YPD-grown gsh1Δ cells re-inoculated in SD medium containing GSH (1 mM) or lacking GSH as indicated, were grown for six divisions, further incubated for 1 h with FeCl₃ (300 μM) or GSH (1 μM) and processed for Leu1 enzyme activity. Results are the mean±s.d. of data from three independent cell cultures. (E) Wild-type (WT) and gsh1Δ petite (rho⁻) cells (Y252 background) depleted of GSH by growth for six divisions in SD medium lacking GSH, serially diluted and spotted onto SD plates containing FeCl₃ (μM) at the indicated concentration, and incubated under aerobic (left panel) or anaerobic (right panel) conditions. (F) GSH non-depleted (grown overnight in SD medium with 1-mM GSH) (1) or depleted (grown for six division in SD medium lacking GSH) (2) gsh1Δ petite (rho⁻) serially diluted and spotted onto SD plates containing DTT (1 mM), GSH (1 mM), FeCl₃ (500 μM) or nothing, and incubated under anaerobiosis.