The Parkin‐siRNA lentivirus and control lentivirus were constructed by Genechem. 3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinoline (DPQ) was from Apexbio (Houston, USA). Cyclosporin A and carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP) was from Selleck. N‐acetyl cysteine was from Beyotime (S0077). Anti‐PAR, Anti‐PARkin, anti‐COX IV, anti‐cyclophilin D (CypD) and anti‐translocator protein (TSPO) was from Abcam. Anti‐poly(ADP‐ribose) polymerase 1 (PARP‐1) was from Proteintech. Secondary antibodies for Western blotting were from Cell Signaling Technology.
N acetylcysteine
Manufactured by Beyotime
Sourced in China, United States
N-acetylcysteine is a chemical compound used in laboratory settings. It is a precursor to the antioxidant glutathione and is commonly used in biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ly294002, by Beyotime (2 mentions)
Bay 11 7082, by Beyotime (2 mentions)
Chloroquine, by Merck Group (2 mentions)
Glucose 6 phosphate (g6p), by Beyotime (2 mentions)
Ldh measurement kits, by Beyotime (2 mentions)
Adenosine triphosphate (atp), by Beyotime (2 mentions)
Pyruvate measurement kit, by Solarbio (2 mentions)
Anti cox 4, by Abcam (1 mentions)
Control lentivirus, by Genechem (1 mentions)
Cyclosporin a, by Selleck Chemicals (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Selleck Chemicals (1 mentions)
Anti par, by Abcam (1 mentions)
Anti parkin, by Abcam (1 mentions)
Secondary antibodies for western blotting, by Cell Signaling Technology (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Hepg2, by National Collection of Authenticated Cell Cultures (1 mentions)
25 protocols using n acetylcysteine
1
Parkin-siRNA Lentivirus Impacts Mitochondrial Function
2
Human Liver Cell Lines Culturing
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Human hepatocellular cell lines Huh-7 and Hep-G2 were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (with 4.5 g/L D-glucose, 584 mg/L L-glutamine, 110 mg/L sodium pyruvate and 3.7 g/L sodium bicarbonate), and supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GEMINI, Woodland, CA, USA) and 1% Pen Strep (Gibco, Waltham, MA, USA). The cells were cultured in an incubator (Thermo HERAcell 150i, Waltham, MA, USA) at 37 °C with a humidified atmosphere of 5% CO2. The PLB compound (2-Methyljuglone, 5-Hydroxy-2-methyl-1,4-naphthoquinone) was purchased from Sigma-Aldrich (CAS 484-42-5, MW = 188.18). PLB was dissolved in dimethyl sulfoxide (DMSO) as a stocking solution at 100 mM. ATM inhibitor KU-55933 (CAS No.:587871-26-9), p53 posttranscriptional activity inhibitor Pifithrin-α (CAS No.:60477-38-5) and ROS scavenger N-Acetylcysteine (NAC, CAS No.: 616-91-1) were purchased from Beyotime Biotechnology (Shanghai, China).
3
Cell Culture and PEDV Strain Propagation
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Vero cells (CCL81) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% FBS (NEWZERUM, Christchurch, New Zealand) and incubated at 37 °C with 5% CO2. LLC-PK1 cells were cultured in DMEM supplemented with 10% FBS and incubated at 37 °C with 5% CO2. PEDV strain DR13 (GenBank accession No. JQ023161) and PEDV strain CV777 (GenBank accession No. AF353511.1) were cultured in Vero cells in DMEM. PEDV strain YN15 (GenBank accession No. KT021228.1) and PEDV strain GDU (GenBank accession No. KU985230) were cultured in Vero cells in DMEM containing trypsin (Gibco, Waltham, MA, USA) at a dose of 8 µg/mL. Mouse anti-PEDV S protein and mouse anti-PEDV N protein monoclonal antibodies were generated in our laboratory [16 (link)]. Rabbit anti-GRP78 polyclonal antibody, Rabbit anti-β-Actin Monoclonal antibody, HRP goat anti-mouse IgG, and HRP goat anti-rabbit IgG were purchased from ABclonal (Wuhan, China). Alexa Fluor 488 Donkey anti Mouse IgG were purchased from AntGene (Wuhan, China). Levistolide A and Tunicamycin were purchased from MedChemExpress (Shanghai, China). DAPI and N-Acetylcysteine were purchased from Beyotime Biotechnology (Shanghai, China).
4
Autophagy Regulation in Cell Assays
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Opti-MEM (31985088) was purchased from Gibco. MitoTracker Red CMXRos (40740ES50) was purchased from YEASEN. DAPI (C1002) and N-acetylcysteine (S0077) were purchased from Beyotime. 3-methyladenine (3-MA) (S2767) and bafilomycin A1 (Baf-A1) (S1413) were purchased from Selleck Chemicals. CCCP (C2759) (SAB5701328) was purchased from Sigma-Aldrich. Anti-Occludin (91131S), Claudin-1 (13255S), GAPDH (5174S), Parkin(2132S), and β-actin (3700S) were purchased from Cell Signaling Technology. Anti-P62 (T55546), ATG5 (T55766), and LC3B primary antibodies were purchased from Abmart.
5
Radiosensitive and Radioresistant NPC Cell Maintenance
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Parental (relatively sensitive to IR, has been previously reported [33 (link)]) and radioresistant subpopulations of NPC cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C and 5% CO2. Cells were plated in 6-well or 12-well plates (Corning Inc., Corning, NY, USA) and the plating efficiencies of cell lines used are higher than 90%. Cells are treated with cycloheximide (CHX; 66-81-9, Abcam, Cambridge, MA, USA), MG132 (S2619, Selleck, Houston, TX, USA), N-acetylcysteine (S0077, Beyotime, Jiangsu, China), or MK2206 (S1078, Selleck) according to studies.
6
Miltirone Inhibits Hepatocellular Carcinoma Viability
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Miltirone (Fig. 1 A) was isolated from the root of S. miltiorrhiza Bunge (S. miltiorrhiza) in our laboratory. The structure was characterized by mass spectrum (MS), 1H nuclear magnetic resonance (NMR), and 13C NMR spectroscopic methods, and the purity of the compound was greater than 98% and re-suspended in dimethyl sulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO, USA). Sorafenib was purchased from Bayer (Leverkusen, Germany). N-Acetyl cysteine (NAC) was from Beyotime Institute of Biotechnology (Nanjing, China). Necrostatin-1 was purchased from Sigma–Aldrich. Ceramide C6 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).Figure 1 ![]()
Miltirone inhibited the viability of HepG2 and Hepa1-6 cells in dose- and time-dependent manners. (A) Chemical structure of miltirone. (B) and (C) HepG2 and Hepa1-6 cells were treated with miltirone (0–80 μmol/L) or Sorafenib (60 μmol/L) for 24 h, cell viability was analyzed by CCK-8 assay and expressed as mean ± SD (n = 3). (D) HepG2 and Hepa1-6 cells were treated with 40 μmol/L miltirone at indicated time, cell viability was analyzed by CCK-8 assay and expressed as mean ± SD (n = 3). ∗∗P < 0.01 vs. control.
7
Cytotoxicity and Oxidative Stress Assays
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T-2 toxin (CAS NO. 21259-20-1) (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium dodecyl sulfonate (SDS), dimethyl sulfoxide (DMSO), chloroquine phosphate salt (CQ) (purity ≥ 99%), and Tris hydroxymethyl (Tris-HCl) were purchased from AMRESCO Inc. (Solon, OH, USA). Rhodamine (Rh) 123, 0.05% Trypsin-EDTA, phenylmethylsulfonyl fluoride (PMSF), 1% (v/v) penicillin and streptomycin, N-acetylcysteine (NAC), and 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) were purchased from Beyotime (Shanghai, China). T-2 toxin was prepared in DMSO at a concentration of 10 μg/mL stock solution and stored at −20 °C. All other reagents were of the highest analytical grade available.
8
Evaluating Mitochondrial Dysfunction in Cells
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Ciclopirox olamine (CPX) was purchased from Dibo Chemical Technology Limited Company (Shanghai, China). BTZ was obtained from Targetmol (MA, USA). Antimycin A, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, oligomycin, and rotenone were purchased from Sigma Aldrich (MO, USA). The Cell Mitochondria Isolation Kit, crystal violet, MTT cell proliferation and cytotoxicity kits, N-acetyl cysteine (NAC), and Nuclear and Cytoplasmic Protein Extraction Kits were purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits were purchased from BD Biosciences (CA, USA), Pierce BCA Protein Assay Kits and TRIzol reagent from Thermo Fisher Scientific (MA, USA), and Protease (cOmplete Mini) and phosphatase-inhibitor cocktail tablets (PhosSTOP) from Roche Applied Science (Penzberg, Germany).
9
Curcumin Inhibits Oxidative Stress in Cells
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CUR (see Fig. 1 for chemical structure) was obtained from BioBioPha Co., Ltd. (Kunming, China). Glucose and mannitol were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 medium with Glucose (5.6 mmol/l) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from HyClone (GE Healthcare, Chicago, IL, USA). Penicillin and streptomycin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). 2′,7′-dichlorodihydrofluororescein diacetate (DCFH-DA), DAPI, LY294002, rapamycin and N-acetylcysteine (NAC) were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Cell Counting Kit (CCK)-8 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). AKT (catalog no. 9272), phosphorylated (p)-AKT (catalog no. 9611) and p-mTOR (catalog no. 2971) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). mTOR (catalog no. 66888-1-Ig) and β-actin (catalog no. 60008-1-Ig) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (catalog no. TA130004) and HRP-conjugated goat anti-rabbit secondary antibodies (catalog no. TA130023) were purchased from the OriGene Technologies, Inc. (Beijing, China).
10
Investigating Nitric Oxide Prodrug JS-K and Antioxidants
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The nitric oxide prodrug JS-K was purchased from Santa Cruz Biotechnology, dissolved in 100% DMSO to a concentration of 5 M as a stock solution, and stored at −20 °C. The final concentration of DMSO did not exceed 0.1% throughout the study. N-acetylcysteine (NAC) and oxidized glutathione (GSSG) were purchased from Beyotime Institute of Biotechnology (Shanghai, China) and dissolved in PBS to concentrations of 100 mM (NAC) and 10 mM (GSSG). Antibodies to poly ADP-ribose polymerase (PARP), caspase-9, Bak, Bcl-2, Bax, cytochrome c, heme oxygenase 1 (HO-1) and apoptosis-inducing factor (AIF) were obtained from Cell Signaling Technology, and an antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Abcam to be used as a loading control. The goat anti-rabbit IgG-HRP secondary antibody was purchased from EarthOx (USA).
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