Nec 1

Carteolol powder (C₁₆H₂₅ClN₂O₃; MW: 328.83 Daltons; CAS Registration No: 51781-21-6; Cat No:BP567, Sigma–Aldrich, St. Louis, MO, United States) with a purity greater than 99.0%, was dissolved and filtered in serum-free DMEM/F12 medium (Cat No: R10-092-CV, Corning, NY, United States), and double-diluted with DMEM/F12 medium that included 20% fetal bovine serum (FBS) (Cat No: 10100147, Gibco, NY, United States), at a final concentration of 0.00390625–2% in DMEM/F12 medium, which included 10% FBS before use in vitro (Shan and Fan, 2016 (link)). The carteolol eye-drop solution was prepared at a clinically relevant therapeutic concentration of 2% by dissolving carteolol power in sterile saline for in vivo experiments. Nec-1 was purchased from MedChem Express (Cat No:HY-15760, NJ, United States), and was dissolved in 50 mg/mL DMSO (Cat No:D2650, Sigma–Aldrich, St. Louis, MO, United States) with sterile saline at a stock concentration of 10 mM, and diluted with sterile saline at a concentration of 10 μM before use. HCECs were cultured in DMEM/F12 medium including 10% FBS, at 37°C in a 5% CO₂ incubator. The HCECs were provided from an established non-transfected HCECs line in our laboratory (Fan et al., 2011 (link)).

Renal cell cancer cell lines 786-O and A498 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, GIBCO, MA, USA) under 5% CO₂ at 37°C. For glucose starvation treatment, cells were cultured in normal medium for 24 h and washed twice with PBS, and the medium was replaced with a glucose-free medium supplemented with 10% FBS.
Cells were treated with the ferroptosis inducer erastin (1.5 μM) (MedChemExpress, China) for 24 h, the ferroptosis inhibitor ferrostatin-1 (2 μM) (Fer-1, MedChemExpress) for 24 h, the AMPK inhibitor Compound C (COM C) (10 μM) (MedChemExpress) for 24 h, the AMPK activator AICAR (2 mM) (MedChemExpress) for 24 h, Pifithrin-α (5 μM) (PFT-α, MedChemExpress) for 24 h, the apoptosis inhibitor Z-VAD-FMK (20 μM) (MedChemExpress), and the necroptosis inhibitor necrostatin-1 (2 μM) (Nec-1, MedChemExpress).

Reagents utilized in this study included: 3-Methyladenine (3-MA; Millipore Sigma, M9281), bafilomycin A1 (Baf-A1; MedChem Express, HY-100558), Chloroquine (CQ; MedChem Express, HY-17589A), Z-VAD-FMK (Z-VAD; MedChem Express, HY-16658B), Necrostatin-1 (Nec-1; MedChem Express, HY-15760), Ferrostatin-1 (Fer-1; MedChem Express, HY-100579), Liproxstatin-1 (Lip-1; MedChem Express, HY-12726) and R162(MedChem Express, HY-103096). Antibodies used in this study were: LC3B (Milliopore Sigma, L7543), SQSTM1/p62 (Milliopore Sigma, P0067), GAPDH (Diagbio, db106), HuR/ELAVL1 (ABclonal Technology, A19622), GLUD1 (Santa Cruz Biotechnology, sc-515542), AGO2 (ABclonal Technology, A19709) and Flag (MultiSciences, 70-AB002-100).

In the first experiment, 36 weanling piglets were randomly divided into six groups including control group and LPS-treated groups slaughtered at five different time points (n = 6). The piglets in LPS-treated groups were injected intraperitoneally (The first pig was injected at 6 a.m.) with Escherichia coli LPS (Escherichia coli serotype 055: B5, Sigma Chemical Inc., St. Louis, MO, USA) at 100 μg/kg body weight (BW), and then were euthanized at 1, 2, 4, 8, or 12 h after LPS challenge. Based on published studies [50 (link),51 (link)] and our preliminary experiments, there was no significant change in inflammation and cortisol of the samples with sham injection at different time points. Thus, the piglets in the control group were euthanized right (0 h) after injection with an equivalent volume of sterile saline. The dose of LPS was administered according to our previous study [52 (link)].
In the second experiment, 28 weanling piglets were randomly divided into four groups including control group, Nec-1 group, LPS group, Nec-1+LPS group (n = 7). Nec-1 (MedChem Express, Monmouth Junction, NJ, USA) was pretreated intraperitoneally (The first pig was injected at 6 a.m.) with 1.0 mg/kg BW or an equivalent volume of 2% DMSO solution 30 min prior to intraperitoneal injection of LPS or saline. The piglets were euthanized at 4 h after LPS or saline injection.

Primary CD4⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, HEPES (10 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM), penicillin, and streptomycin. Naïve CD4⁺ T cells were isolated from spleens and lymph nodes of 8-week-old WT and Ddb1^fl/fl; OX40-cre mice, and cells were co-cultured with Antigen Presenting Cells (APC cells) in 96-well U bottom plate with soluble 1 μg/ml anti-mouse CD3 and 1 μg/ml anti-mouse CD28 antibodies. CD4⁺ T cells were activated for 0–6 days and harvested for flow cytometry analysis or western blotting.
Experiment to restore the phenotype of Ddb1 deficient activated CD4+ T cells and CD4⁺ T cells were treated with various inhibitors in CD4⁺ T-cell stimulation assay. Z-VAD (cat #HY-16658B), Z-IETD (cat #HY-101297), Ferrostatin-1 (cat # HY-100579), GSK872 (cat #HY-101872), Nec1 (cat # HY-15760), AZD7762 (cat # HY-10992), and Rabusertib (cat # HY-14720) were purchased from MedChemExpress; Nec-1s (cat #S8641) was purchased from Selleck.