Mitoq

U2OS, A549, Saos-2, and HEK293 cells (purchased from ATCC) were cultured either in Dulbecco’s modified Eagle medium (Sigma, D5796) or Roswell Park Memorial Institute Medium (RPMI)-1640 supplemented with 10% fetal bovine serum (Thermo Scientific, E6541L), 2 mml-Glutamine (LabClinics, M11–004) and 100 μg/ml penicillin–streptomycin (LabClinics, P11–010). For the inhibition of p38α, we used 1 μm PH797804 (Selleckchem, S2726) or 500 nm BIRB0796 (Axon MedChem, 1358), and 200 nm LY2228820 (Selleckchem, S1494). MK2 was inhibited using 10 μm MK2 Inhibitor III (Calbiochem, 475864–5MG) or 10 μm PF 3644022 (Sigma, PZ0188). The following additional treatments were used: 0.5 μm MitoQ (kindly provided by M. Murphy, Cambridge, UK), 250 nm Doxorubicin hydrochloride (Sigma, D1515–10mg), 50 μm Z-VAD (OMe)-FMK (SM Biochemicals LLC SMFMK001), 200 or 50 nm bafilomycin A1 (Santa Cruz, sc-201550), 20 μm chloroquine (Sigma, C6628), 10 μm Spautin-1 (Axon MedChem, 2512), 15 μm palbociclib (Selleckchem, S1116), 500 nm camptothecin (Sigma, C9911). The compounds were dissolved in dimethyl sulfoxide (DMSO) or water and the total concentration of DMSO in the culture medium never exceeded 1%.

The septic ALI model was induced by cecal ligation and puncture (CLP). Briefly, animals were fasted for 12 hours before surgery. The rats in CLP group, MitoQ group, and MitoQ + LY294002 group were anesthetized with pentobarbital sodium (50 mg/kg). The abdomens of rats were shaved, and their cecums were exposed by a 2 cm abdominal midline incision. The cecum was isolated and ligated below the ileocecal valve, punctured by using a 21-gauge needle. Next, faeces were squeezed through the puncture wound. Finally, the cecum was returned to the peritoneal cavity and abdominal incision was then sutured. Rats in the Con group were only treated with laparotomy and distal cecum isolation. After operation for 0 h and 6 h, rats in the Con group and the CLP group were treated with normal saline (1 mL) by intraperitoneal injection, respectively. Meanwhile, rats in the MitoQ group were treated with MitoQ (2 mg/kg, Cell Signaling, USA) by intraperitoneal injection. In addition, rats in MitoQ + LY294002 group were intraperitoneally injected with MitoQ (2 mg/kg) and LY294002 (20 μmol/L, Sigma, USA), respectively. After CLP treatment, the survival of the rats was recorded at 0, 3, 6, 12, and 24 h, and the survival rate was calculated.

C2C12 skeletal myoblast cells (ATCC CRL-1772, USA) were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, NY, USA) supplemented with fetal bovine serum (FBS) to a final concentration of 10%. The cells were passaged every second day, and confluency was maintained below 80% to prevent spontaneous differentiation. First, 5,58,6,68 tetraethylbenzimidazolcarbocyanine iodide (JC-1), EX-527, 2',7' –dichlorofluorescein diacetate (DCF-DA) and MitoSox reagent were obtained from Thermo Scientific (Waltham, MA, USA). MitoQ, SB203580, penicillin, and streptomycin were purchased from Sigma (St. Louis, MO, USA). Anti-β-actin, anti-p38, anti-p-p38, anti-NF-kBp65, anti-HDAC1, and anti-caspase-3 antibodies were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-AMPK, anti-SIRT1, anti-PGC-1α, anti-Bax, anti-Bcl2, HRP-conjugated anti-rabbit, and anti-mouse secondary antibodies were purchased from Transduction Laboratories (CA, USA).

Paraquat (PQ)(Sigma Aldrich) was diluted from a 0.1 M stock prepared with ultrapure water (Invitrogen). Antibodies for western blotting and immunocytochemistry: rabbit anti‐catalase (#12980), rabbit anti‐VDAC (#4866) and rabbit anti‐Caspase 3 (#9662) from Cell Signaling; rabbit anti‐catalase (sc‐50508), mouse anti‐p53 (sc‐126) and rabbit anti‐Nrf2 (sc‐722) from Santa Cruz; mouse anti‐ATP5α (ab14748) and rabbit anti‐catalase (ab16731) from Abcam; rabbit anti‐phosphoNrf2ser40 (bs‐2013R) from Bioss; mouse anti‐ PKCδ (cat #36520) from Transduction Laboratories; mouse anti‐Tubulin (ATN01) from Cytoeskeleton. Inhibitors: sodium azide from Fisher Scientific; PKCδ (Gö6983), PKCα/β (Gö6976) and Caspase 3 (Z‐DEVD‐FMK) from Calbiochem. Fluorescent Dyes: propidium iodide (PI), 2′,7′‐dichlorodihydrofluorescein diacetate (DCF), tetramethylrhodamine methyl ester perchlorate (TMRM) from Molecular Probes. Mitochondrial targeted antioxidants: MitoQ, 1 mM stock in H₂O; and MitoTEMPO, 2 mM stock in H₂O, from Sigma‐Aldrich. Nrf2 activators: Oltipraz (Tocris) 5 mM stock in DMSO; Sulforaphane (Cayman Chemical), 5 mM stock in ethanol.

OLI-neu, an immortalized oligodendrocyte cell line, was kindly gifted by Professor Lan Xiao (Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University) and cultured in DMEM (Gibco, USA) with 10% fetal calf serum (FCS; Gibco, USA), N2 supplement (Gibco, USA), and insulin (Sigma, USA). For the cell death experiment and mitochondrial membrane potential, the OLI-neu cells were exposed to 250 μM FeCl₂ for 48 hours with or without 400 nM MitoQ and 1 mM NAC (N-acetyl-L-cysteine; Sigma, USA). For ATP and mitochondrial ROS assays, OLI-neu cells from each group were treated as described above for 24 hours.