ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death

Figure 4

Implication of the necroptosis pathway in iron-induced osteoblastic necrosis. (a) Total protein levels of phosphorylated RIPK1, RIPK3, MLKL, and corresponding total protein. After exposure to FAC (50-200 μM) for 120 h, western blot was used to detect the expression of phosphorylated RIPK1, RIPK3, MLKL, and corresponding total protein in osteoblastic cells. (b) Histogram analysis showing the relative protein levels of phosphorylated RIPK1, RIPK3, MLKL, and corresponding total protein. Data are presented as the from three independent experiments ( vs. saline control, ANOVA test). (c) Representative data were analyzed using the flow cytometer after Annexin-V/PI staining in osteoblastic cells after exposure to 50-200 μM FAC for 120 h. (d) Histogram statistical analysis demonstrating the ratio of PI positive cells. Values are expressed as the from three independent experiments ( vs. saline control, ^# vs. FAC 200, ANOVA test). (e) The protective effects of Nec-1, GSK872, or NSA on the osteoblastic cell viability were evaluated by the CCK-8 assay. Values are expressed as the from three independent experiments ( vs. saline control, ^# vs. FAC 200, ANOVA test).

(a)

(b)

(c)

(d)

(e)