24-Epibrassinolide, a Phytosterol from the Brassinosteroid Family, Protects Dopaminergic Cells against MPP+-Induced Oxidative Stress and Apoptosis
Figure 7
(a) Histogram showing the percentage of apoptotic cells counted on triple-stained slides (merged), as described in Materials and Methods. To illustrate that caspase-3 activation is a key step in the MPP+-induced apoptotic pathway, PC12 neuronal cells were pretreated with Z-DEVD-FMK, a cell-permeable caspase-3 inhibitor, followed by treatment with MPP+. Pretreatment with 24-Epi strongly reduced the number of apoptotic neuronal PC12 cells induced by treatment with MPP+ (24-Epi + MPP+), and pretreatment with the caspase-3 inhibitor prevented MPP+-induced apoptosis. The data are expressed as percentages of apoptotic cells and are means ± S.E.M. . and versus Ctrl and versus MPP+. (b) Immunofluorescence detection of apoptotic neuronal PC12 cells on triple-stained slides (merged), as described in Materials and Methods. Blue: neuronal PC12 nuclei were counterstained in blue with DAPI. Red: anticleaved caspase-3 antibody. Green: TUNEL. Merged: cells are considered apoptotic when positive for both TUNEL (green) and anticleaved caspase-3 antibody (red). They appear as light blue cells (arrows) because of the third superposition of dark blue DAPI. Triple-staining (merged) clearly reveals several apoptotic cells, indicated by arrows, on slides treated with MPP+ (MPP+) and fewer apoptotic PC12 cells (arrow) when they are pretreated with 24-Epi prior to MPP+ addition.
