Necrostatin 1 nec 1

Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO, USA), a source of iron, was utilized to mimic iron overload conditions in vivo and in vitro [7 (link), 9 (link), 22 (link)]. To evaluate the cytotoxic effect of iron overload, the MC3T3-E1 osteoblastic cells were exposed to FAC (50, 100, and 200 μM) for 24, 72, and 120 h, respectively. The control groups were treated with 0.9% saline solution. According to our previous experiments, the cell viability of osteoblastic cells significantly decreased from 24 to 120 h [9 (link)]. More importantly, with iron overload exposure time prolonged, the osteoblastic cell necrosis peaked at 120 h. Thus, 120 h FAC-treated time periods were chosen throughout the following study. N-acetyl-cysteine (NAC) (Beyotime Biological Technology, Shanghai, China) was dissolved in phosphate-buffered saline (PBS). The RIPK1 inhibitor Necrostatin-1 (Nec-1) (Selleck, Houston, TX), MLKL inhibitor Necrosulfonamide (NSA) (Selleck, Houston, TX), and RIPK3 inhibitor GSK872 (Selleck, Houston, TX) were dissolved in DMSO solution. Before exposure to FAC, the MC3T3-E1 osteoblastic cells were incubated with or without NAC (1 mM), Nec-1 (20 μM), GSK872 (4 μM), or NSA (4 μM) [9 (link), 23 (link)–25 (link)]. Then, after FAC (200 μM) treatment for 120 h, all samples were collected and analyzed by a microplate reader, flow cytometry, western blots, and confocal microscopy.

Antibodies against Drp1, p62, LC3, RIP1, RIP3, TSG101, and CD63 were purchased from Abcam (Cambridge, MA, USA). β-actin, tubulin, and COX IV, which were used as references for the total, cytoplasmic, and mitochondrial fractions, were also acquired from Abcam (Cambridge, MA, USA). Antibodies against Phospho-RIP3 (Thr231/Ser232) and Phospho-Drp1 (Ser616) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Aggresome Detection Kit was acquired from Abcam (Cambridge, MA, USA). MitoTracker Deep Red, the ROS 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, dihydroethidium (DHE) assay kit, and MitoSOX detection kit were purchased from Invitrogen (Carlsbad, CA, USA). Ad-mRFP-GFP-LC3 was supplied by Beyotime (Shanghai, China). Adenoviral vectors for Drp1 short hairpin RNA (shRNA) and Drp1 mutations (Drp1-S616D and Drp1-S616A) were generated by Genechem Technology (Shanghai, China). Related activators and inhibitors, including Mitochondrial division inhibitor 1 (Mdivi-1), N-acetylcysteine (NAC), and Necrostatin-1 (Nec-1), were obtained from Selleck (Shanghai, China). The Protein A/G Magnetic Beads IP Kit was purchased from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS) and penicillin/streptomycin were procured by Invitrogen (Carlsbad, CA, USA). All other chemicals were supplied by Sigma unless otherwise specified.