Figure 3.

Caspase-3 activation is inhibited by Hsp70. Motoneurons were plated with or without trophic support (MEx) or without MEx but treated with recombinant human Hsp70 (75 μg/ml) for 24 h and fixed and stained with anti-cleaved caspase-3. Phase-contrast (A-C) and fluorescent (D-F) images are presented. Results are expressed as a percentage of positively labeled cells in each condition. As expected, cultures with no MEx showed significantly more activated caspase-3-immunoreactive cells compared with cultures with MEx (G; ^*p < 0.001). Cultures with e-rhHsp70 showed a significant reduction in staining for activated caspase-3 compared with cultures without MEx (G; ^#p<0.001) and no difference compared with controls with MEx. Statistical significance was determined by ANOVA (n = 3 independent experiments with 3-4 wells per condition per experiment; error bars represent SEM). H, Although caspase-3 activation was decreased in motoneurons deprived of MEx, but treated with Hsp70, we found no direct inhibition of activity by Hsp70. Hsp70, ovalbumin control protein, or the caspase inhibitor, DEVD, were added at equimolar amounts (0.7 μm). Readings were taken every 2 min up to 10 min, and then every 15 min up to the 90 min time point. ABS, Absorbance.