Fig. 5.
Inhibition of glutathione synthesis but not Hsp70/Hsc70 activity abolishes stress-induced protection against the 2nd MG132 hit. A) Primary cortical astrocytes were treated with the indicated concentrations of MG132 in the absence or presence of the Hsp70/Hsc70 inhibitor VER155008 and glutathione synthesis inhibitor buthionine sulfoximine (BSO). Viable Hoechst+ nuclei were quantified 24h after the 2nd hit to measure viability. B) Data from panel A were expressed as a percentage of the 0 μM 2nd MG132 hit groups (i.e. all gray bars were expressed as a percentage of the adjacent black bars). C) Representative images of Hoechst-stained nuclei from data shown in panel A. D) Total glutathione levels were measured 24h after the 2nd MG132 hit by the In-Cell Western technique and expressed as a function of the corresponding number of Hoechst+ nuclei to control for differences in cell density. E) Representative image of total glutathione In-Cell Western. F–H) Reduced glutathione levels were measured intracellularly and in the extracellular media by the Glutathione-Glo assay and luminescence was expressed as a function of the number of Hoechst+ nuclei on parallel plates. All glutathione assays in F–H were performed 24h after the final MG132 hit. I–J) Western blot analysis of glutamate cysteine ligase modifier subunit (GCLM) and glutathione S-transferase μ (GST-μ) in lysates collected 24h after the 1st MG132 hit. K) Astrocytes were treated with single and dual hits of MG132 in the absence or presence of BSO and ATP levels were measured by the CellTiter-Glo assay 24h after the 2nd MG132 hit. ATP levels are expressed as a function of Hoechst+ cell numbers on parallel plates. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 μM 2nd MG132 hit; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 vs 0 μM 1st MG132 hit; ^ p ≤ 0.05, ^^ p ≤ 0.01 vs 0 μM VER155008 and/or 0 μM BSO, two- or three-way ANOVA followed by Bonferroni post hoc correction. For F–G and I–J, the two-tailed paired Student’s t test was employed