Z vad

HK-2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI 1640 media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA) in an incubator system (humidified atmosphere of 5% CO₂ at 37 °C; Thermo Fisher Scientific Inc.). Cells were treated with doxorubicin (DOX; 1 μM; Sigma-Aldrich Co. LLC, St. Louis, MO, USA), etoposide (ETO; 50 μM; Sigma-Aldrich Co. LLC) and z-VAD (25 μM; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Treated and untreated HK-2 cells were harvested using trypsin-EDTA (Thermo Fisher Scientific Inc.) for 3 min in a humidified atmosphere with 5% CO₂ at 37 °C.

Nec-1 was prepared in DMSO at a concentration of 1 μg/3 μl,^{27 (link)} and z-VAD (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) was prepared in DMSO at a concentration of 100 μM.^{10 (link)} At 1–2 h before ICH, both the inhibitors were injected into the lateral cerebral ventricle at a volume of 3 μl. Equal volumes of DMSO were used as vehicle. For in vitro experiments, the TNF-α inhibitor (Santa Cruz Biotechnology) was also dissolved in DMSO at a final concentration of 50 μM in a neuronal medium. The final concentration of Nec-1 and z-VAD was 30 and 100 μM, respectively, in a neuronal medium.

Poly(I:C) and Poly(dA:dT) (InvivoGen), puromycin (Gibco), Lipofectamine 2000 and Lipofectamine RNAiMAX (Invitrogen), Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology), MG132 (Sigma‐Aldrich), LPS (Sigma‐Aldrich), zymosan (InvivoGen), β‐glucan (Curdlan AL, InvivoGen), imiquimod (Invitrogen), CpG ODN2395 (Invitrogen), Sepharose 6B (GE Healthcare Life Science), glutathione‐conjugated Sepharose 4B (GST) beads (GE Healthcare Life Science), Strep‐Tactin Sepharose (1201‐0590, IBA), NH₄Cl (Sigma‐Aldrich), Z‐VAD (Santa Cruz Biotechnology), chloroquine (Sigma‐Aldrich), and LysoTracker Red DND‐99 (Thermo Fisher Scientific) were obtained commercially. All the antibodies used for the immunoblot analysis and immunofluorescence experiments are listed in Appendix Tables S1 and S2.

H9c2 cells were treated with SUN (1.2 μM), LAP (1.5 μM), 5FU (120 μM), or CDDP (10 μM) concurrent with the following cell death inhibitors for 24 h: ZVAD (10 μM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NAC (1 mM) (Sigma), DFO (100 μM) (Cayman Chemicals, Ann Arbor, MI, USA), FRS (1 μM), and 4PBA (20 μM).

The following primary antibodies were used: anti-MHC (MF20, Developmental Studies Hybridoma Bank, 1:1,000), anti-active-caspase 3 (ab13847, Abcam, 1:500), anti-cleaved caspase 3 (9661, Cell Signaling, 1:500), anti-YFP (ab6673; Abcam, 1:1,000), anti-RFP (600-401-379, Rockland, 1:500), anti-myc (05-724; Millipore, 1:100), anti-P53 (sc-99, sc-6243, Santa Cruz, 1:200), anti-MDM2 (sc-965, Santa Cruz, 1:200), anti-P19 (ab80, abcam, 1:500) and anti-GAPDH (AM4300, Invitrogen, 1:1,000). Appropriate horseradish peroxidase-conjugated and Alexa Fluor-conjugated secondary antibodies were used for western blot and immunofluorescence studies. The following reagents were used: Staurosporine (BD Biosciences) was used in concentrations ranging between 0.005 and 1 μM. Myoseverin (Sigma) was used at 25 μM. 4,4′ diisothiocyanatostilbene-2,2′-disulphonic acid (Molecular Probes) was used at 100 μM. TMRE (Invitrogen) was used at 10 nM. Z-VAD (Santa Cruz) or Q-VD-OPH (Santa Cruz) was used at 10 μM. YO-PRO-1 (Molecular Probes) was used at 1 mM. FLIVO tracer was from ImmunoChemistry Technologies. TUNEL assay was performed with In Situ Cell Death Detection Kit (Roche). EdU staining was performed by incubating 30 min with 100 mM Tris, 1 mM CuSO₄, 10 μM fluorescent azide and 100 mM ascorbic acid43 (link).

Nec-1, NAC, WM, and BAPTA were purchased from Merck (Darmstadt, Germany). Annexin V-FITC/PI and JC-1 dye were from Abcam (Cambridge, UK). MitoSOX and MitoTracker Red FM were from Thermo Fisher (Waltham, MA, USA). Antibodies against PARP, caspase-3, β-actin, α-tubulin, p38, p-JNK, p-ERK, IκBα, c-FLIP, tBid, Bcl-xL, Bcl-2, Bax, and Mcl-1 were obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated mouse anti–human IgG (Fab specific) Ab and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). The z-VAD was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and dissolved in dimethyl sulfoxide. sTRAIL and DR4/DR5 were purchased from Koma Biotech (Seoul, Korea) and Strathmann Biotec (Hamburg, Germany), respectively. Human DcR1/Fc chimera and human DcR2/Fc chimera were from R&D Systems (Minneapolis, MN, USA).