Ldh detection kit

Cells with different treatments were collected and incubated for 2 h in medium supplemented with 150 μl of lactate dehydrogenase (LDH) release reagent. After 5 min of centrifugation, concentrations of LDH in the supernatants were measured according to the standard protocols of a commercial LDH Detection Kit (Beyotime).

PS NPs and NH₂-PS NPs were acquired from Sigma–Aldrich (Sigma-Aldrich, St. Louis, MI, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein di-acetate (DCF), JC1, and LDH Detection Kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). TRIzol reagent was acquired from Takara (Takara Biochemicals, Dalian, China). The ReverTra Ace^® qPCR RT Kit and SYBR^® Green Realtime PCR Master Mix were from Toyobo (Tokyo, Japan). All oligonucleotide primers were synthesized in Sangon Biotech (Sangon, Shanghai, China).

As previously reported, the MTT assay (Beyotime) was used to determine cell (NRCMs) viability. According to the manufacturer's instructions, cell damage is assessed by determining the release of LDH from the cell using the LDH detection kit (Beyotime).

3-(3,4-Dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), brusatol (BT, an Nrf2 inhibitor), and dimethylsulfoxide (DMSO) were obtained from Sigma Aldrich (St Louis, MO, USA). The Bicinchoninic acid protein H₂O₂ assay (BCA) kit, RIPA lysis buffer, ROS detection kit, and LDH detection kit were obtained from Beyotime Biotechnology (Shanghai, China). α-Cyperone (> 98% purity; Yuan ye Biotech, Shanghai, China) was dissolved in DMSO and freshly diluted to the final concentration of 0.05% DMSO. The Annexin V-FITC/PI Apoptosis Detection Kit was obtained from Solarbio (Beijing Solarbio Science & Technology, Beijing, China). Phosphate buffered saline (PBS), Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Grand Island, NY, United States). Primary antibodies against Nrf2, Bax, Bcl-2, cleaved-caspase-3, PCNA, and β-actin were obtained from Proteintech Group (Wuhan China). Secondary goat anti-rabbit or goat anti-mouse antibodies were obtained from Santa Cruz, CA, USA.

Esculetin was purchased from Yuanyebio (Shanghai, China). Anisomycin (Ani), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from Solarbio (Beijing, China). A LDH Detection Kit and a JC-1 Staining Kit were purchased from Beyotime (Shanghai, China). An Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Key gen Biotech (Nanjing, China). DMEM was purchased from Sigma Aldrich (St. Louis, IL, USA). Fetal bovine serum was purchased from ExCell Bio (Taicang, China). Antibodies against Bax, Bcl-2, Caspase-1, and p-c-Jun were purchased from Proteintech (Chicago, MO, USA). Antibodies against cleaved-Caspase-3, and p-c-Fos were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against c-Fos, c-Jun, Caspase-3, JNK, and PARP were purchased from Beyotime (Shanghai, China). Anti-β-actin antibody was purchased from Solarbio (Beijing, China). Antibodies against cleaved-PARP, Cytochrome c (Cyt-c), GSDMD, IL-1β, NLRP3, and p-JNK were purchased from Abcam (Cambridge, UK).