Figure 3. Dual hits of proteotoxic and oxidative stress are synergistic 48h after the final insult.

Primary hippocampal cultures were treated on DIV5 with the 1^st hit of MG132 and on DIV6 with the 2^nd hit of paraquat. Viability was assayed 48h later on DIV8 by (A) the nuclear DRAQ5 stain, (B) the In-Cell Western assay for MAP2 levels, and (C) the Cell Titer Glo luminescent assay for ATP. Representative DRAQ5 and MAP2 images are shown in panels G and H. Panels A–C are shown as scatterplots in Supplemental Figure 3 (A–C) to show all individual data points. (D–F) The data shown in panels A–C were expressed as a function of the 0 μM paraquat 2^nd hit group (i.e., all gray bars were expressed as a percentage of the adjacent black bars). The latter measurements show statistically that the 1^st hit significantly exacerbates the toxic impact of the 2^nd hit on DIV8. Shown are the mean and SEM of 4 independent experiments, each performed in triplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μM 1^st hit; +p ≤ 0.05, ++p ≤ 0.01, +++p ≤ 0.001 versus 0 μM 2^nd hit; two-way ANOVA followed by Bonferroni post hoc correction.