To assess the effect of BSO (Selleckchem), NAC (Selleckchem), or cell death inhibitors including ferrostatin-1 (Selleckchem) and Z-VAD-FMK (Selleckchem) on the efficacy of olaparib (Selleckchem), cells with appropriate density per well were incubated in triplicate in 6-well or 12-well plates for 24 h. Cells were then treated with DMSO, BSO, NAC, olaparib, or olaparib in combination with BSO or NAC or indicated cell death inhibitors at appropriate concentrations for 48 h. olaparib was then removed, and the medium was replaced every 48 h with fresh medium containing DMSO, BSO, NAC, or indicated cell death inhibitors. Cells were stained with 0.5% crystal violet (Sigma) dissolved in 20% methanol following 1–2 weeks of incubation, after which the number of colonies were counted visually (or crystal violet was redissolved in methanol and then absorbance was measured at 570 nm). The survival fraction was calculated using GraphPad Prism 6 and normalized to that of control (DMSO) cells.
N acetyl cysteine (nac)
Manufactured by Selleck Chemicals
Sourced in United States
NAC is a laboratory reagent used as a source of the compound N-Acetylcysteine. It is a white crystalline powder with a purity of typically 98% or greater. NAC is widely used in various chemical and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Z vad fmk, by Selleck Chemicals (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ferrostatin 1, by Selleck Chemicals (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Selumetinib, by Selleck Chemicals (2 mentions)
3 typ, by Selleck Chemicals (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
Oxaliplatin, by Selleck Chemicals (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Melatonin, by Merck Group (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Fluorouracil, by Selleck Chemicals (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
22 protocols using n acetyl cysteine (nac)
1
Evaluating Olaparib Efficacy Modifiers
2
Ang II-Induced Renal Tubular Epithelial Cell Responses
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Human renal proximal tubular epithelial HK-2 cells (Cell Bank of Shanghai Biology Institute, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco) in an incubator (37 °C, 95%, CO2:5%). The cells in the vehicle group were cultured in DMEM + vehicle. Cells in the Ang II group were cultured in DMEM with +1 µM Ang II. Cells in the N-acetylcysteine (NAC) group were cultured in DMEM + 1 µM Ang II + 100 µM NAC (Selleck, Radnor, PA, USA), which was used as a positive control. The cells in the BSHX group were cultured in DMEM +1 µM Ang II + BSHX (0.1, 0.2, or 0.5 mg/mL). Finally, cells were collected for further assays after 24 h of treatment.
3
Cell Signaling Pathway Analysis
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Fluorouracil, selumetinib, BIO and NAC were purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO or diluted water. DCF-DA was purchased from the Life Technology (Invitrogen, Carlsbad, California, USA) and dissolved in DMSO. Melatonin was from Sigma Aldrich (Sigma-Aldrich, St. Louis, USA). Antibodies include PARP, pAkt, Akt, pErk, Erk, GSK3β, pGSK3β, MEK, pMEK, β-Actin and GAPDH (Cell Signaling Technology, Beverly, MA, USA) as well as Ki-67 (Abcam, Cambridge, Massachusetts, USA).
4
ROS Accumulation Measurement Protocol
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A Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology) was used to detect ROS accumulation. According to the manufacturer's instructions, the cells were pre-treated with or without N-acetylcysteine (NAC; 5 mM, Selleck Chemicals) or glutathione (GSH; 1 mM, GlpBio) for 1 h at 37°C prior to the addition of Cu(sal)(phen) and harvested (200 × g, 5 min, 4°C), followed by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 20 min at 37°C. Samples were detected, and the data were analyzed using a BD Accuri™ C6 Flow Cytometer (BD Biosciences) and FlowJo 10.6.2 software (BD Biosciences), respectively.
5
Preparation and Storage of Chemical Solutions
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The chemicals BBM (MedChemExpress, HY-N0714), BAPTA-AM (MedChemExpress, HY-100545), and rifampicin (MedChemExpress, HY-B0272) were dissolved in dimethyl sulfoxide (DMSO), while 3-MA (MedChemExpress, HY-19312), NAC (Selleck, S1623), CQ (Cell Signaling, 14774s), and Isoniazid (MedChemExpress, HY-B0329) were dissolved in double-distilled water. All solutions were aliquoted and stored at −20°C or −80°C.
6
Ubenimex Induces Apoptosis in Cancer Cells
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Ubenimex were provided by Shenzhen Main Luck Pharmaceuticals, Inc. (Shenzhen, China). LIVE/DEADTM Cell Imaging Kit and Total Reactive Oxygen Species (ROS) Assay Kit were purchased from Thermo Fisher Scientific (USA). Cell Counting Kit-8 was purchased from Dojindo (Japan). Annexin V-FITC/PI kit was purchased from BD Biosciences (USA). NAC, 3-MA and Z-VAD-FMK were purchased from Selleck (USA). Primary antibodies: caspase-3 (1:500; catalog # ab13847), parp1 (1:2000; catalog #ab32138), cleaved parp1 (1:2000; catalog #ab32064) and β-actin (1:5000; catalog # ab8226) were purchased from Abcam (UK). ERK (1:1000; catalog # 4695T) and p-ERK (1:1000; catalog # 4370T) were purchased from Cell Signaling Technology (USA). Caspase-9 (1:1000; catalog # 10380-1-AP) was purchased from Proteintech (USA).
7
Comprehensive Autophagy and Signaling Pathway Analysis
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The antibodies used in our experiments were: LC3 (CST, #2775), IL-33 (R & D, AF3626 and AF4810), ST2 (Thermofisher, PA5-20077), p-STAT3 (CST, #9145), STAT3 (CST, #9139), p-AMPK (CST, #2535), AMPK (CST, #2532), p-ULK1 (Thermofisher, PA5-105129), ULK1 (Abclonal, A8529), p62 (Abclonal, A1970), GKN-1 (Proteintech, 19344-1-AP), GKN-2 (Abcam, ab188866), GKN-3 (Cloud-clone, PAK528Mu01), Tubulin (Proteintech, 66031-1-Ig).
The chemicals used in our experiments were: Bafilomycin A1 (Selleck, S1413), 3-MA (Selleck, S2767), Rapamycin (Selleck, S1039), MNNG (Selleck, E0157), NAC (Selleck, S1623), Cycloheximide (Selleck, S7418), DAPI (Beyotime, C1002), Recombinant Human IL-33 (R & D, 3625-IL), Pifithrin-α HBr (Selleck, E0157), 2-MeOE2 (Selleck, S1233), ML385 (Selleck, S8790), SR11302 (MCE, HY-15870), BAY 11 (Selleck, S2913), GW9662 (Selleck, S2915), IQ3 (Selleck, S0781), STAT3-IN-7 (Selleck, S0986), FH535 (Selleck, S7484).
The chemicals used in our experiments were: Bafilomycin A1 (Selleck, S1413), 3-MA (Selleck, S2767), Rapamycin (Selleck, S1039), MNNG (Selleck, E0157), NAC (Selleck, S1623), Cycloheximide (Selleck, S7418), DAPI (Beyotime, C1002), Recombinant Human IL-33 (R & D, 3625-IL), Pifithrin-α HBr (Selleck, E0157), 2-MeOE2 (Selleck, S1233), ML385 (Selleck, S8790), SR11302 (MCE, HY-15870), BAY 11 (Selleck, S2913), GW9662 (Selleck, S2915), IQ3 (Selleck, S0781), STAT3-IN-7 (Selleck, S0986), FH535 (Selleck, S7484).
8
Modulation of N. caninum Infection in Macrophages
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PMs were isolated from WT or Nlrp3−/− mice and infected with N. caninum at a multiplicity of infection (MOI) of 3:1, 2:1 or 1:1 (parasite: cell) in R-1% medium for 2, 4 or 8 h, then PMs were washed twice with PBS to remove non-adhered parasites. In the experimental group, cells were pre-treated with the ROS inhibitor NAC for 1 h (5 mM; Selleck, Shanghai, China), or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, 10 μM; Selleck) for 2 h [30 (link)]. Additionally, at 2, 4 or 8 h post-infection (pi), the medium was changed to fresh medium with or without the ROS inducer pyrogallol (PG; Selleck). PMs cultured with the equivalent volume of R-1% medium was used as a negative control. Adenosine triphosphate (ATP) is a NLRP3 inflammasome inducer [31 (link)], so PMs in positive control group were pre-treated with N. caninum and then stimulated with 5 mM ATP (Sigma-Aldrich, Shanghai, China) for 30 min. At 5, 24 or 36 h post-infection, cells and supernatants were collected for subsequent experiments, as described below.
9
Investigating Apoptosis and Signaling Pathways
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Ara-C and DNR were purchased from the Sigma-Aldrich (St. Louis, MI, USA). NAC, Momordin-Ic and 3-TYP were consumed from Selleckchem (Houston, TX, USA). All compounds, except for in vivo studies, were reconstituted in the DMSO, stored at 100 mM stock concentrations in −80 °C, and used at the indicated doses as suggested by the vendor. Flow cytometry antibodies, Alexa Fluor 647 Rabbit anti-Active caspase 3 and APC-H7 Mouse anti-Human CD45 were purchased from BD pharmingen (San Jose, CA, USA). PE/Cy5 anti-Mouse CD45 (clone 30-F11) was consumed from BioLegend (San Diego, CA, USA). Immunoblotting antibodies, SUMO1, SIRT3, SENP1, Notch Activated Targets Antibody Sampler Kit including Notch1 (FL), MAML1, BPUSH and HES1, p-PI3K p85, t-PI3K p85, p-p38, t-p38, p-AKT and t-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA). Acetylated SOD2 and SOD2 antibodies were purchased from Abcam (London, UK). Tubulin and β-actin antibodies were purchased from Proteintech (Rosemont, IL, USA).
10
Evaluation of Apoptosis and Oxidative Stress
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Anti‐Myc (#sc‐40) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti‐HIF‐1α (#36169) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐β‐actin (#AC026) was purchased from ABclonal Company (Wuhan, China). Dual‐luciferase reporter assay system (#E194A) was purchased from Promega (Madison, WI, USA). 3‐TYP (#S8628) and NAC (#S1623) were purchased from Selleck (Houston, TX, USA). FITC‐Annexin V Apoptosis Detection Kit I (#556547) was purchased from BD Pharmingen (San Diego, CA, USA). CM‐H2DCFDA (#C6827) and MitoSOX™ Red (# M36008) were purchased from Thermo Fisher (Waltham, MA, USA). Annexin V‐FITC Apoptosis Detection Kit (#C1062) was purchased from Beyotime (Shanghai, China).
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