Fig. 2.

U73122‐induced apoptosis and necroptosis in IL‐1β‐treated rat chondrocytes, and the effect of combined treatment with the apoptosis and necroptosis inhibitors Z‐VAD and Nec‐1, respectively. Rat chondrocytes pretreated with IL‐1β (20 ng·mL⁻¹ for 36 h) were treated with U73122 (2 μm for 12 h). Cell proliferation (A), percentage of dead cells (B), and ROS level (C) were measured. Protein levels of apoptosis and necroptosis indexes Bcl‐2, Bax, P53, pro/cleaved‐caspase3, RIP1, and RIP3 were analyzed by Western blotting (D). Chondrocytes pretreated by IL‐1β (20 ng·mL⁻¹ for 36 h) and U73122 (2 μm for 12 h) were treated with Z‐VAD (10 μm for 12 h) or/and Nec‐1 (30 μm for 12 h). Cell proliferation (E), percentage of dead cells (F), and ROS level (G) were measured, and protein levels of apoptosis and necroptosis indexes were analyzed by Western blotting (H). The results of ROS were normalized, means of the first group in Fig. 2C,G were used as the control for ROS. β‐Actin was used as the control for Western blotting. Values are means and standard deviations, the error bars represent SD. One‐way ANOVA with the Dunnett test was used to calculate P values. These results are representative of at least three independent experiments in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.