Figure 4

Lower level of glutathione and YAP1 gene expression exacerbates oxidative stress in eIF5^G31R mutant cells. (a) Glutathione quantification. Yeast strain YP823 carrying EV pYcplac22 (A823) or pYcplac22-eIF5^G31R (A838) constructs were grown in SD plus leucine, uracil medium till OD₆₀₀ ~ 0.8 at 30 °C. 200 μl cells suspension were stained with 30 μM of monochlorobimane (mCBM) followed by fluorescence microscopic imaging at Em λ₄₆₀. The values are average (n = 300) along with the standard error of the mean (SEM). (b) Growth analysis in the presence of glutathione. Yeast strains as per (a) were spotted on either SD plus leucine, uracil plus 2 mM H₂O₂ or supplemented with 5 mM glutathione plates and incubated at 30 °C for 3 days. (c) Quantification of YAP1-LacZ expression. Yeast strain YP823 carrying EV pYcplac22 (A823) or pYcplac22-eIF5^G31R (A838) constructs were transformed with pYCplac111-YAP1_LacZ (A1311) or pYCplac111-YAP1-upTTGless_LacZ (A1312) construct, were grown till OD₆₀₀ ~ 0.8 at 30 °C and the β-galactosidase activity was measured as per Fig. 2b. The error represents an average deviation. (d) Growth analysis in the presence of H₂O₂. Yeast strain YP823 carrying EV pYcplac22 (A823) or pYcplac22-eIF5^G31R (A838) constructs were transformed with either high copy EV pRS425 (B1378) or (h.c) pRS425-YAP1 construct (A1346), grown overnight, serially diluted, and spotted on SD, uracil plus 2 mM H₂O₂ as per Fig. 3c. Statistical differences were determined by the two-tailed Student’s t-test. ns non-significant.