Tcmk 1

SVEC4-10 (CRL-1658), NIH-3T3 (CRL-1658), TCMK-1 (CCL-139), M2-10B4 (CRL-1972), and HEK 293T (CL-11268) were obtained from the ATCC. HEK-293A cells (R705-07) were purchased from Invitrogen. Murine 10.1 fibroblasts have been described [69 (link)]. Immortalized murine bone marrow-derived macrophages (iBMDM) were obtained from BEI Resources, NIAID NIH (NR-9456). BMDM expressing firefly luciferase under control of the endogenous IFN-β promoter were isolated from transgenic mice [70 (link)] and immortalized by transduction with retrovirus J2 as described [71 (link)]. Ifnar^-/- iBMDM have been described [45 (link)]. All cells were cultured at 37°C and 5% CO₂ in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and 100 IU penicillin/100 μg streptomycin.

Human renal carcinoma (Caki, ACHN, and A498), human breast carcinoma cells (MCF-7), human lung carcinoma cells (A549), normal human umbilical vein cell (EA.hy926), and normal mouse kidney cells (TCMK-1) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 20 mM HEPES buffer, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml gentamicin. The PCR primers were purchased from Macrogen (Seoul, Korea). Recombinant human TRAIL and BIX were purchased from Sigma Chemical Co. (St. Louis, MO, USA). z-VAD-fmk and anti-survivin antibodies were purchased from R&D system (Minneapolis, MN, USA). Anti-Mcl-1 and anti-cIAP2 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Bim and anti-XIAP antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-PARP, anti-Bcl-2, anti-Bcl-xL, anti-DR5, and anti-G9a antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-actin antibody and other chemicals were purchased from Sigma Chemical Co.

Human renal tubular epithelial HK-2 cells and mouse kidney epithelial cell line (TCMK-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; 10564029) with 10% fetal bovine serum (Gibco; 16000044) and 100 U/mL penicillin and streptomycin (Solarbio, Beijing, China; P1400-100). HK-2 cells were treated with 5.5 mM normal glucose (NG) and 30 mM high glucose (HG) for 48 h. The NG group was additionally treated with 24.5 mM mannitol to establish the control osmolarity.

HK2, an immortalized proximal tubule epithelial cell line from the adult human kidney, was purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, P.R. China). The mouse tubular epithelial cell line TCMK-1 (#CCL-139TM) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in RPMI1640 medium (Wisent Corporation, Nanjing, China) containing 10% fetal bovine serum (FBS, Gibco Corporation, USA) and 1 × 10⁵ U/L streptomycin sulfate (Gibco) in a constant environment of 37° C with 5% CO₂. The cells were then incubated with Fru in the absence or presence of CA (HPLC ≥ 95%, Sensient Technologies, Guangzhou, China). The negative control (NC) and Nrf-2-specific siRNAs (siNrf-2) were synthesized and purchased from Shanghai Generay Biotech (Shanghai, China) and transfected into cells using Lipofectamine® 3000 (Invitrogen, USA) according to the manufacturer’s protocols. In order to induce inflammation and fibrosis in vitro, lipopolysaccharide (LPS; #L8880, Solarbio, Beijing, China) and Recombinant Human TGF-β (#P00121, Solarbio) or Mouse TGF-β (#P00199, Solarbio) were exposed to cells.

Mouse renal tubular epithelial (TCMK-1) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The TCMK-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) that was supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) in a 5% CO₂ humidified atmosphere at 37 °C.

The human renal proximal tubular cell line HK-2 and mouse kidney epithelial cell line TCMK-1 were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (ThermoFisher, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a 37 °C incubator with humidified air containing 5% CO₂. For the construction of I/R model in vitro, hypoxia/reoxygenation (H/R) of HK-2 cells and TCMK-1 cells was used to mimic the I/R in vitro as described in the previous study^{32 (link)}. HK-2 cells were exposed to hypoxia (1% O₂, 5% CO₂, and 94% N₂) for 24 h followed by 12 h of reoxygenation (21% O₂, 5% CO₂, and 74% N₂). For the cell transfection, cells were transfected with either miR-182-5p and miR-378a-3p mimic or inhibitor (Ribobio, Guangzhou, China) by using Lipofectamine 3000 reagents (ThermoFisher, USA) or treated with GPX4 lentivirus or SLC7A11 lentivirus (Genepharma, Shanghai, China) for 24 h before the H/R treatment. The sequences of the mimic or inhibitors used were as follows: miR-182-5p mimic (sequence: 5′-UUUGGCAAUGGUAGAACUCACACU-3′), inhibitor (sequence: 5′-AGUGUGAGUUCUACCAUUGCCAAA-3′); miR-378a-3p mimic (sequence: 5′-ACUGGACUUGGAGUCAGAAGG-3′), inhibitor (sequence: 3′-CCUUCUGACUCCAAGUCCAGU-3′).