Fig. 2.
MG132-mediated severe proteotoxic stress preconditions astrocytes against subsequent insults. A) Primary cortical astrocytes were treated with indicated concentrations of the proteasome inhibitor MG132 one day after plating (1st hit) or an equal v/v of vehicle (dimethyl sulfoxide). Twenty-four hours later, astrocytes were challenged with a 2nd hit of MG132 (3.125 μM) or vehicle. Astrocyte viability was assessed 24h after the 2nd hit by counting viable Hoechst+ nuclei in blinded fashion. B) Data in panel A were expressed as a percentage of each 0 μM 2nd MG132 hit group (i.e., each gray bar was expressed as a percentage of the adjacent black bar). C) Representative images of Hoechst-stained astrocyte nuclei from the groups shown in A. The interval between the 1st hit and the 2nd hit was prolonged to 48h in D–F and to 96h in G–I. E and H illustrate data in D and G, respectively, expressed as a percentage of the 0 μM 2nd MG132 hit group. Representative Hoechst-stained nuclei are shown in F and I. J–K) The interval between the 2nd hit and the viability assay was extended to 72h. The data in panel J were expressed as a percentage of the 0 μM 2nd MG132 hit group and presented in panel K. L) Representative images of Hoechst-stained nuclei from groups shown in J. * 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, two-way ANOVA followed by Bonferroni post hoc correction