Fig. 3

Necroptosis of mouse spinal cord astrocytes in vitro. a, b Western-blotting of RIP3 and MLKL in control, TLZ (TNFα, LPS and Z-VAD), TLZN (TLZ plus Nec-1). Notice that TLZ significantly increased the expression of RIP3 and MLKL. This effect was significantly suppressed by Nec-1. **P <0.01, *P <0.05. n = 3. c, d Western-blotting of cytoplasmic and nuclear HMGB1 in control, TLZ or TLZN-treated astrocytes. Notice that TLZ significantly increased cytoplasmic HMGB1, but decreased nuclear HMGB1. The effects of TLZ on HMGB1 were blocked by Nec-1. e ROS staining in control, TLZ treated, TLZN treated and RIP3^−/− astrocytes. f PI-staining in control, TLZ treated, TLZN treated and RIP3^−/− astrocytes. g, h Quantification of intracellular ROS and ATP levels in astrocytes under various conditions. i Percentages of PI-positive cells in TLZ treated, TLZN treated astrocytes and TLZN treated RIP3^−/− astrocytes. Bars in (e), F = 50 μm. **P <0.01. *P <0.05. n = 3