Fig. 7.
Stressed astrocytes prevent the synergistic, severe toxicity of proteasome and Hsp70/Hsc70 inhibitors in stressed neurons. A) Astrocytes were treated with the 1st MG132 hit or vehicle 24h prior to introduction of neocortical neurons, in the absence or presence of the Hsp70/Hsc70 inhibitor VER155008. Two days after the introduction of neurons, the neuron/astrocyte co-cultures were treated with vehicle or MG132 as a 2nd hit in the absence or presence of VER155008. Two days after the 2nd hit, neuronal viability was measured by the In-Cell Western assay for MAP2. B) Data from panel A were expressed as a percentage of the 0 μM VER155008 groups to illustrate the exacerbation of MG132 toxicity by VER155008. C) Representative image of MAP2 In-Cell Western. D) The astrocytic marker glial fibrillary acidic protein (GFAP) was analyzed by In-Cell Western analyses 48h after the 2nd hit in the same neuron/glia co-cultures as shown in panels A–C. E) Representative image of GFAP In-Cell Western. * p ≤ 0.05, *** p ≤ 0.001 vs 0 μM 2nd MG132 hit; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 vs neurons; ^ p ≤ 0.05, ^^ p ≤ 0.01, ^^^ p ≤ 0.001 vs 0 μM VER155008, three-way ANOVA followed by Bonferroni post hoc correction