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Alpha-mangostin (99% purity), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC), and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1), were purchased from Sigma (St. Louis, MO, USA). Z-VAD, a pan-caspase inhibitor, was obtained from BioVision (Mountain View, CA, USA). The kinase inhibitors, PD98059 to MEK, SB203580 to p38, and SP600125 to JNK, were bought from Calbiochem (San Diego, CA). The antibodies against phosphory-ERK, phosphory-p38, phosphory-JNK, ERK, p38, JNK, phosphory-MKK3/6, MKK3/6, Bcl-2, Bax, cytochrome C, COX4, α-tubulin, β-actin, siRNA-ERK, siRNA-JNK and siRNA-p38, siRNA-ASK1, siRNA-MKK3/6 were purchased from Santa Cruz Biotechnology (Dallas, Texas). The antibodies against cleaved-caspase-3, cleaved-caspase-9, cleaved-poly-ADP-ribose polymerase (PARP), phosphory-ASK1 and ASK1 were purchased from Cell Signaling (Danvers, MA, USA). Horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies were bought from Promega (Madison, WI, USA).

To test whether silencing A20 could counteract the anti-necroptotic role of Nec-1 and melatonin, SD rats were randomly assigned into six groups: sham+AAV-shCtrl group (n = 25), CCI+AAV-shCtrl group (n = 27), CCI+Nec-1+AAV-shCtrl (n = 27), CCI+Melatonin+AAV-shCtrl (n = 27), CCI+Nec-1+AAV-shA20 (n = 27), CCI+Melatonin+AAV-shA20 (n = 27). Tissue samples were collected at 6 h except for behavioral test (Supplementary Figure S1D). A schedule for AAV administration and drug treatment for experiment design 4 was displayed in Supplementary Figure S1E.
Melatonin (N-acetyl-5-methoxytryptamine, #M5250) was dissolved in 5% ethanol in saline (20 mg/kg) and injected into the left side of the peritoneum at 1 h before the CCI (Wong et al., 2014 (link)). Nec-1 (Selleck, Houston, TX, USA) and Z-VAD (BioVision, Mountain View, CA, USA) were dissolved in 10% dimethyl sulfoxide (Sigma-Aldrich). Rats was administered Nec-1 or Z-VAD via intracerebroventricular injection (coordinates: 0.8 mm posterior, 1.5 mm right lateral, and 4.0 mm ventral from Bregma). Infusion of 2 μg Z-VAD in 10 μl vehicle was conducted at a rate of 20 μl/h for 30 min (Li et al., 2014 (link)). Six microliter Nec-1 with concentration of 25 mM was used at a rate of 0.5 μl/min (Liu et al., 2016 (link)). In this study, experimenters made efforts to minimize the pain of rats.

LNCaP and PC3 PCa cells, original from American Type Culture Collection (ATCC), were gifted by Dr. Mu Yao and Prof. Thomas Grewal at the University of Sydney, Australia, respectively. The cell cultures were maintained at 37 °C in a humidified incubator with 5% CO₂. All PCa cell lines were cultured in phenol red-free Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Erianin, bafilomycin A1 and S-trityl-L-cysteine (STLC) were purchased from Sigma, while z-VAD was obtained from BioVision. All treatments were dissolved in dimethyl sulfoxide (DMSO). PC3 cells were transfected with CerS5 and CerS6 plasmids (Genscript) using lipofectamine LTX Plus reagents (Thermo Fisher).