Figure 7. Rad6 supports translation and cellular resistance to stress.

(A) Immunoblot of WT and rad6Δ cells incubated in the presence or absence of 0.6 mM H₂O₂ for 15 min, following 30 min of puromycin incorporation (0.9 mM) prior to cellular harvesting. Anti-GADPH was used as loading control.

(B) Fluorescence determination of GFP in WT and rad6Δ cells. GFP expression was under a Met25 promoter and was induced at time zero by transferring cells to Met-depleted medium. *p < 0.05.

(C) Growth curve of WT and rad6Δ strains in the presence (dashed) or absence (solid) of H₂O₂.

(D) Reactive oxygen species (ROS) determination by 2.5 μM CellRox Deep Red fluorometric assay (Thermo Fisher Scientific) from WT and rad6Δ cells exposed to 0.6 mM H₂O₂ for 30 min. Fluorescence was captured at 665 nm with excitation at 644 nm. Graph shows mean ± standard deviation for three biological replicates; p values were determined by Student’s t test.

(E) Volcano plot of SILAC proteomics ratio from H₂O₂-treated and untreated (unt) WT and rad6Δ cells (0.6 mM H₂O₂ for 30 min). Proteins with 1.5× fold change and p <0.05 across three biological replicates are highlighted in the graph.

(F) Heatmap of MS/MS intensity fold change for antioxidant proteins detected in the WT and rad6Δ cells.

(G) Immunoblot anti-myc for the expression of the antioxidant proteins Tsa2, Gpx2, and Sod2 in WT and rad6Δ cells. Cells were treated with 0.6 mM H₂O₂ for the respective times denoted in the figure. Anti-actin was used as loading control.

(H) Bar graph of qPCR analysis of TSA2, GPX2, and SOD2 transcript levels relative to TAF10 from untreated and H₂O₂-treated WT and rad6Δ cells (0.6 mM H₂O₂ for 30 min). Graphs show mean ± standard deviation for three biological replicates.