Figure 1
ESA induces apoptotic cell death in neuronal cells. (a) PC12, SH-SY5Y, NG108-15 and FBD-102b cells, as well as rat cortical neurons were used to observe morphological changes of ESA-mediated apoptosis. Cells were cultured under a differentiation condition (in the case of cell lines) and exposed to 2 μg/ml ESA for several hours in cell lines and 48 h in rat neurons. ESA initiated membrane blebbing and formation of apoptotic bodies in neuronal cells (inset in each figure). The time course of morphological changes in PC12 cells is shown at the bottom, indicating clear membrane blebbing at 4 h after ESA treatment. Nucleus of PC12 cells treated with DMSO or ESA were shown in blue (Hoechst 33342 staining). (b) Rat neurons cultured in B27-supplemented media survived, whereas the cells in antioxidant-deficient B27-AO-supplemented media were killed by ESA (20 μg/ml) for 48 h. Values are the means±S.D. (n=3; *P<0.05 to DMSO control). Dendrites of rat neurons were stained with anti-MAP2 antibody (red). ESA disrupted the dendrites. (c) ESA-mediated apoptosis is AIF/RIP1-dependent and RIP3-independent in neuronal cells. Cells transfected with pcDNA, pcDNA-bcl-2, or pcDNA-bcl-XL were exposed to ESA. Bcl-2 and Bcl-XL did not block ESA-mediated apoptosis. Knockdown of AIF and RIP1 blocked ESA-mediated apoptosis. Double knockdown (AIF/RIP1) had no synergistic effect against ESA-mediated apoptosis. Cell viability of RIP3-knockdown cells is the same as non-target control (NT) when stimulated with ESA. RIP3 protein was hardly expressed in PC12 cells. Western blot data of cells knocked down with AIF, RIP1, and RIP3 were shown. NT, non-target control in siRNA experiments. (d) Nec1 blocked ES-mediated apoptosis, whereas Nec5 did not. Nec1 inactive control, which cannot block necroptosis, inhibited ESA-mediated apoptosis.
