Figure 1.
Comparison of the sensitivity of strains T73 and T73trx2Δ to different stressful conditions.
Growth of strains T73 and T73trx2∆ indicated on YPD (A) or YPG plates (B) containing no chemicals as a control, 5 mM H2O2, 0.1 mM menadione, 1.2 diamide, 16.5 % ethanol. For each strain, 5 µL of the overnight culture diluted to 1.0 OD600nm units and seven subsequent dilutions (10-1 dilution factor) were spotted on plates. Growth was monitored after 2 days for YPD plates and 3 days for YPG plates. (C) The growth rate for strains T73 and T73trx2∆ over 25 h in distinct carbon source media. (D) Viable cells after 1 h exposure to different H2O2 doses in various media. Three independent experiments were carried in all conditions.
Figure 2.
Antioxidant gene expression levels in strains T73 and T73trx2∆ cultured in YPD or YPG medium to the mid-log phase and treated with 5 mM H2O2 for 60 min.
The relative expression levels of each antioxidant gene were quantified from the Northern image analysis using the Image Gauge software (FujiFilm, USA). The data for each gene were normalized to the rRNA levels. Experiments were carried out in triplicate and the error bars corresponding to SD are shown. Statistical analyses were performed using a Student´s t-test between samples in the same growth media, YPD or YPG (* p< 0.05, ** p< 0.01). The Yap1p and the Yap1p-Skn7p-dependent gene groups were formed according to the previously described information [21, 22, 43] and from the Yeastract web site (http://www.yeastract.com).
Figure 3.
Promoter regulation by the Yap1p and Skn7p transcriptional factors in strains T73 and T73trx2∆.
(A) The cells carrying the depicted modification of the TRX2 promoter fused to the LacZ vector construction (T73-YRE and T73trx2∆-YRE) for Yap1p recognition or (B) the cells carrying the depicted modification of the GPX2 promoter fused to the LacZ vector construction (T73-GCRE and T73 trx2∆-GCRE) for Yap1p-Skn7p recognition were cultured in YPD (C, D) or YPG medium (E, F) until the mid-log phase and were treated with different H2O2 concentrations (0.4, 1, 2.5 and 5 mM) for 60 min. Experiments were carried out using three biological replicates and the error bars corresponding to SD are shown. Statistical analyses were performed using a Student´s t-test between samples in the same growth media, YPD or YPG (* p< 0.05, ** p< 0.01).
Figure 4.
The expression analysis of the heat shock protein genes after exposure to oxidative stress conditions.
(A) Northern blot analysis of HSP12, HSP26, HSP70 and HSP104 in strains T73 and T73trx2∆ grown on YPD or YPG to the mid-log phase and treated with 5 mM H2O2 for 60 min. (B) Relative expression levels were quantified by an image analysis. The data for each gene were normalized to the expression levels of the control strain in YPD medium under non stressed conditions. Three independent experiments were carried and the error bars corresponding to SD are shown. Statistical analyses were performed using a Student´s t-test between samples in the same growth media, YPD or YPG (* p< 0.05, ** p< 0.01).
Figure 5.
The Skn7p phosphorylation pattern after exposure to different H2O2 concentrations.
(A) Cells from the T73-SKN7 (myc13x) control strain grown in YPD medium until the mid-log phase were exposed to 0.4 mM for 60 min. Lambda phosphatase (Ph) and phosphatase inhibitors (Iph) were added to the protein extracts as indicated and samples were incubated for 30 min at 30°C, following the manufacturer´s recommendations. Skn7p-myc western blotting was performed after protein separation in 8% SDS-PAGE. (B) The T73-SKN7 (myc13x) strain was cultivated in YPD and YPG medium until the mid-log phase and cells were exposed to 0 (-), 0.4 mM (+) and 5 mM (++) H2O2 for 1 h. (C and D) The Skn7p phosphorylation pattern in the TRX2 gene-modified strain under oxidative stress, 0.4 mM and 5 mM H2O2 for 60 min. (D) The protein extracts from the cells grown on YPD and exposed to 0.4 mM H2O2 for 1 h were treated with lambda-phosphatase, as indicated. (E) Analysis of the different post-transcriptional modifications using Skn7-Myc-tagged immunoprecipitation. Cells were cultivated in YPD medium and 0.4 mM H2O2 was added for 1 h. Representative experiments from five replicates are shown. A dashed gray line was added to the figure based on the migration of the untreated wild-type samples for each analyzed gel.