N acetylcysteine nac

Pancreatic cancer cells were incubated for 72 h in 60 mm
² culture dishes with glutamine-free medium, medium containing 2 mM glutamine, or medium containing 2 mM glutamine supplemented with 50 μM diazooxonorleucine (Cat. #HY-108357; MedChemExpress, Shanghai, China) or 10 μM azaserine (Aza; Cat. #HY-B0919; MedChemExpress). Then, the cells were incubated with 10 μM 2’,7’-DCFH-DA (Cat. #D6883; Sigma-Aldrich, St Louis, USA) in serum-free DMEM at 37°C for 30 min. Cells treated with 100 μM H
₂O
₂ were used as positive controls. Thereafter, cells were treated with glutamine-free medium containing 2 μM ferrostatin-1 (Cat. #HY-100579; MedChemExpress), 5 mM N-acetylcysteine (NAC, Cat. #HY-B0215; MedChemExpress), 50 μM Z-VAD-FMK (Cat. #HY-16658B; MedChemExpress), and 150 μM necrostatin-1 (Cat. #HY-15760; MedChemExpress) to counteract cell death. The cells were then washed twice, resuspended, and filtered into single-cell suspensions before analysis. The fluorescence intensity of DCFH, formed by the reaction of DCFH-DA dyes with ROS, was measured at excitation and emission wavelengths of 488 and 530 nm, respectively, with a FC500 MPL flow cytometer (Beckman Coulter, Pasadena, USA). A minimum of 10,000 cells were analyzed per condition
[13] (link). Proportion analysis and histogram generation were performed using FlowJo software (version 10.6; FlowJo, Ashland, USA).

MC3T3-E1 mouse pre-osteoblast cell line was bought from the Shanghai Cell Center. The passage numbers of cell lines used for the experiments were from 2 to 10. MC3T3-E1 cells were seeded at the density of 4 × 10⁵ cells/well in a six-well polystyrene plate. Cells were cultured in the phenol red-free alpha-minimum essential medium (a-MEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillin/streptomycin. Cultures were maintained at 37 °C in a fully humidified atmosphere of 5% CO₂ in air. The culture medium was changed every 3 days. 17b-estradiol (E₂; Sigma-Aldrich, St. Louis, MO, USA) was added at a concentration of 10⁻⁷ M. N-Acetyl Cysteine (NAC; MedChem Express, USA) was added at a concentration of 30mM. The concentration was chosen based on the manufacturer’s recommendation and previously published studies. Following initial cell seeding, cells were incubated for 24 h. Then cells were cultured in phenol red-free a-MEM without FBS for 12 h to eliminate the estrogenic action before being pretreated with vehicle or reagents for 1 h15 (link)16 (link). Cells were then treated with vehicle or 0.2 mM H₂O₂ 20 min for real time-PCR, or 30 min for cell immunofluorescence and Western blot analysis.

ISL was purchased from Selleck Chemicals (Shanghai, China), and cisplatin was obtained from Sigma-Aldrich (St. Louis, MO, United States). Antibodies against cleaved caspase-3 and caspase-3 (19677-1-AP), Bax (50599-2-Ig), Bcl-2 (26593-1-AP), β-actin and horseradish peroxidase-conjugated goat anti-mouse antibody (SA00001-1) were purchased from Proteintech Group (Rosemont, IL, United States). Antibodies against SOD2, NF-κB (p-65) and Phospho-NF-κB (Pp-65) were purchased from Cell Signaling Technology (Beverly, MA, United States). Horseradish peroxidase-conjugated goat anti-rabbit antibody and the reactive oxygen species (ROS) assay kit were purchased from Beyotime Biotech (Nantong, China). An apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, United States). The CCK-8 assay kit was purchased from ApexBio (MA, United States). N-Acetylcysteine (NAC) was purchased from MedChemExpress. All other reagents were of reagent grade or better, and deionized water was used for all the experiments.

AF, ICG-001, and N-acetyl cysteine (NAC) were purchased from MedChemExpress Inc. (Monmouth Junction, NJ). For co-treatment, the cells were incubated with AF and ICG-001 premixed under the dose ratio of 1:10 (2 μM AF, 20 μM ICG-001, 2 μM AF + 20 μM ICG-001) for 24 h; for the experiments regarding the N-acetyl cysteine (NAC) inhibition, the cells were pre-treated with 5 mM NAC dissolved in culture medium for 1 h before drug treatment (17 (link)); for the mice co-treatment, mice were treated with AF and ICG-001 premixed under the dose ratio of 1:2 (5 mg/kg AF, 10 mg/kg ICG-001, 5 mg/kg AF + 10 mg/kg ICG-001) for 21 days (39 (link)).

The NPCs were cultured in conventional medium (control group, 330 mOsm/kg) or hyperosmotic medium (experimental group, 550 mOsm/kg). The conventional medium is prepared by adding 10% fetal bovine serum (Gibco, Grand Island, New York, USA) and 1% penicillin/streptomycin (Beyotime, Wuhan, China) to DMEM/F-12 medium (Hyclone, Logan, Utah, USA). The hyperosmotic medium was obtained by addition of NaCl based on the conventional medium. Endoplasmic reticulum stress inhibition: rat NPCs were treated with sodium phenylbutyrate (4-PBA, 2.5 nmol/L, MedChemExpress, Monmouth Junction, New Jersey, USA) in DMEM/F-12 medium for 30 min, then change to hyperosmotic medium for 24 h. ROS inhibition: rat NPCs were treated with N-acetylcysteine (NAC, 1 μmol/L, MedChemExpress, Monmouth Junction, New Jersey, USA) in hyperosmotic medium for 24 h.

Sources and cultures of human normal bronchial epithelial BEAS-2B cells and alveolar type II epithelial A549 cells have been described in detail in our previous research [11 (link)]. IGF1R was silenced in 40 passages of AECs induced by CD-NPs (IGF1R-siRNA) with use of knockout lentiviruses according to the manufacturer’s instructions (Sangon Biotechnology Co., Ltd., Shanghai, China).
Picropodophyllin (PPP, AXL1717) (a selective IGF-1R inhibitor), MK2206 (a specific inhibitor of AKT) and TWS119 (a specific inhibitor of GSK3β) were procured from Selleck Chemicals (Houston, USA). Recombinant human IGF1 was obtained from PeproTech (New Jersey, USA). N-Acetylcysteine (NAC) (a ROS inhibitor) and MCC950 (a potent and selective NLRP3 inhibitor) were bought from MedChemExpress (New Jersey, USA).

Cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol)2000] (DSPE-PEG-2000), Cy5.5-DSPE-PEG and dipalmitoyl phosphatidylcholine (DPPC) were obtained from Xi’an Ruixi Biological Technology Co., Ltd. Protoporphyrin IX (PpIX), 3-Methyladenine (3-MA) and N-acetylcysteine (NAC) were purchased from MedChemExpress. 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was bought from Beyotime Biotechnology.2,2,6,6-Tetramethylpiperidine(TEMP),3,8-diamino-5- [3-(diethylmethylammo nio) propyl] -6-phe-nylphenanthridinium diiodide (PI), 3′,6′-di(O-acetyl) -4′,5′-bis [N, N-bis(carboxymethyl)aminomethyl] fluorescein, and tetraacetoxymethyl ester (Calcein-AM) were purchased from Dojindo Molecular Technologies. MitoScreen JC-1 Kit was bought from BD Pharmingen. Cell-counting kit-8 (CCK-8) and DAPGreen (D676) autophagy detection probe were purchased from Dojindo Molecular Technologies.