Fig. 3.

15d-PGJ₂ induces primary neuronal cell death via a caspase-3 independent mechanism. Cells were treated with vehicle (Veh, methyl acetate), CAY10410 (CAY), 15d-PGJ₂ for 24 h – 96 h. Pro-apoptosis reagents, MG132 and staurosporin, were incubated with primary neurons at 10 μM for 16 h as positive controls. (a). Caspase-3 and PARP cleavage: Cells were harvested and immunoblotted using anti-cleaved caspase-3 and anti-PARP antibodies. β-actin was used as a loading control. Full: full length protein; Clvd: cleaved. (b). Representative immunocytochemical photos of rat primary neurons after treatment with Veh or 2.5 μM 15d-PGJ₂. Upper panel: Cleaved caspase-3 was detected using anti-cleaved caspase-3 antibody (green) and anti-NeuN (red) antibodies (48 h after treatment). Middle panel: Activated caspase 3/7 was detected using a fluorogenic substrate for activated caspases-3 and 7 (green, 72 h after treatment). Lower panel: Double-stranded DNA breaks were detected using TUNEL (green, 72 h after treatment). All photos: 60X using a confocal microscope; scale bar = 40 μm. Blue is DAPI nuclear stain