N acetyl cysteine (nac)

BC-23 (NSC45382) was obtained from the NCI database. ICRT14, N-acetyl cysteine (NAC), and H₂DCFDA, were purchased from Santa Cruz Biotechnology, Sigma-Aldrich, and Cayman-Chemical, respectively. H1299 and H1975 cells were purchased from ATCC. The cells were cultured and maintained in a humidified 5% CO₂ atmosphere at 37°C in RPMI1640 (Hyclone, Thermal Scientific) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin.

All treatments were performed in RPMI containing HEPES. Sub-confluent cells were treated with SSZ (Sigma-Aldrich, Antwerp, Belgium, S0883) overnight (16 h). ROS scavenger, N-acetyl cysteine (NAC) (10 mM) (Santa Cruz Biotechnology, Heidelberg, Germany, 616-91-1), was added to cultures 1 h prior to and during treatment with SSZ. Ferroptosis inhibitor Ferrostatin-1 (5 µM) (SanBio, Uden, The Netherlands, 17729) was added during treatment with SSZ.

The isolated cells were seeded at a density of 1 × 10⁶ cells per well in a six-well plate in RPMI-1640 with 10% fetal bovine serum (HyClone, UT, USA), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Beyotime, China) at 37°C and 5% CO₂. Cells were grown to 80–90% confluence. For heat stress treatments, culture dishes were placed into incubators with different temperatures (39 ± 0.5°C, 41 ± 0.5°C, and 43 ± 0.5°C) for different times (0, 15, 30, 45, and 60 min) and a culture humidity >90%. Control cells were maintained in incubators at 37 ± 0.5°C.
To block caspase-1, the caspase-1 inhibitor AC-TYR-VAL-ALA-ASP-chloromethylketone (ac-YVAD-cmk) (0.3 mg/ml; Enzo Biochem, Inc., New York, USA) was added to the cells for 30 min before heat stress. MCC950 is a specific inhibitor of NLRP3, and MCC950 (50 μM, MedChemExpress, Shanghai, China) dissolved in normal saline was given 1 h before heat stress in vitro. Z-DEVD-FMK (3 μM, Sigma-Aldrich, USA) as the specific caspase-3 inhibitor was added to the cells 1 h before heat stress. In some experiments, the antioxidant N-acetyl cysteine (NAC, Beyotime, 5 mM) and the mitochondria-targeted antioxidant Mito-TEMPO (1 μM, Santa Cruz Biotechnology) were added to the cells before heat stress.

Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N-acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] was obtained from Sigma-Aldrich (St. Louis, MO, USA). Enzalutamide (ENZ) was purchased from ApexBio (Houston, TX, USA). Cycloheximide (CHX) was bought from Cayman chemicals (Ann Arbor, MI, USA). CDDO-Me was purchased from Selleckchem (Houston, TX, USA). The drugs were dissolved in 100% DMSO and the final DMSO concentration which was used in the experiments was less than 0.1%. N-acetyl cysteine (NAC) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in water and diluted in media immediately before use. The primary antibodies including rabbit polyclonal anti-AR (N-20) (sc-816), mouse monoclonal anti-Nrf2 (437C2a) (sc-81342), and anti-GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology. The horseradish peroxidase (HRP)-conjugated goat anti-rabbit (A0545) and goat anti-mouse (A9044) secondary antibodies were bought from Sigma-Aldrich (St. Louis, MO, USA). The goat antirabbit secondary antibody tagged with Texas red (T-2767) was bought from Thermo Scientific.