Every green

The mouse umbilical cord mesenchymal stem cell line mUC-MSC (BNCC340370) and mouse mesangial cell line SV40-MES-13 (ATCC^CRL-1927) were purchased from BeNa Culture Collection (BeNa Chuanglian Biotechnology Research Institute, Beijing, China) and American Type Culture Collection (Manassas, VA 20108, USA), respectively. Both cell lines were cultured in DMEM medium (HyClone, Thermo Fisher Scientific, Logan, Utah, USA) supplemented with 10% FBS (EVERY GREEN, Zhejiang Tianhang Biotechnology Co. Ltd., Zhejiang, China), 100 U/mL penicillin, and 100 μg/mL streptomycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in a humidified atmosphere of 5% CO₂ at 37°C.
Some of the SV40-MES-13 cells were placed in 6-well plates and cultured with normal glucose (NG, 5.6 mM) or high glucose (HG, 30 mM) for 24 hours. Meanwhile, the other SV40-MES-13 cells were seeded in the transwell chamber and then placed on a 6-well plate that contained mUC-MSCs. After coculturing for 24 hours, the SV40-MES-13 cells were harvested for further analyses.

Two RAS‐driven LUAD cell lines (A549 and NCI‐H1299) were purchased from the Chinese Academy of Science Cell Bank (https://www.cellbank.org.cn/). The cell lines were authenticated by short tandem repeat (STR) profiling before use. Cells were cultured in the DMEM (High Glucose), supplemented with 10% fetal calf serum (Every Green, Zhejiang Tianhang Biotechnology), as well as penicillin/streptomycin/amphotericin B (Beyotime Biotechnology). Cells were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.
They were transfected by lentivirus containing green fluorescent protein (GFP), and untransfected cells were filtered out by flow cytometry. Then GFP‐included cells were transfected with siRNAs at a 100 nM final concentration using the Lipofectamine8000 (Beyotime) and the Opti‐MEM (Thermo Fisher Scientific). siRNAs were designed by and purchased from the Guangzhou RiboBio Co, China. We used two different siRNAs separately targeting each gene (IRX2, SPINK13, and CAPN8) and two different siRNAs without targeting the human genome as controls. The targeting sequences of each siRNA can be accessed in the Table S2. The efficiency was assessed using quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis after transfection. Primers used can be obtained in the Table S3.

SHSY5Y cells were obtained from ATCC (ATCCCRL-2266) and grown in DMEM/F12 medium (hyclone) supplemented with 10% FBS (EVERY GREEN, Zhejiang Tianhang Biotechnology Co., Ltd, China), 100 μg/mL streptomycin, and 100 U/ml penicillin (Beijing solarbio science & technology co., Ltd) in high humidity condition with 5% CO₂ at 37°C. After culturing the cells in 100 mm dishes to reach a ~70% confluence, they were subjected to hydralazine, H₂O₂ or MPP⁺ treatment. The dose and duration of application of hydralazine, H₂O₂ or MPP⁺ are provided in the figures and text. SiRNA interference were performed by treating the cells with Nrf2 SiRNA (sc-37030) or control SiRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA) in 6-well plates for 24 h using the Lipofectamine 3000 reagent (Thermo Fisher Scientific Co., Carlsbad, CA, USA) as indicated in the instructions provided by the manufacturer. After transfection for approximately 24 h, SHSY5Y cells were exposed to hydralazine with or without MPP⁺. After these treatments, cells were used for biochemical analysis.

MC3T3-E1 cells (MC3T3-E1 Subclone 14) and C3H/10T1/2 (Clone 8) were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and maintained in α-MEM supplemented with 100 U/mL penicillin–streptomycin (Beyotime) and 10% fetal bovine serum (FBS, Every Green, ZheJiang TianHang Biotechnology Co., Ltd., Hangzhou, China). Bone marrow stromal cells (BMSCs) were harvested and cultured as reported [37 (link)]. Briefly, long bones were first separated from C57BL/6J mice aged 2–3 months. Then, small cuts were made at both the proximal and distal ends of the bones, and a syringe was used to flush the cells. The cells were collected, centrifuged, and suspended in α-MEM with 10% FBS and 1% penicillin–streptomycin, then plated in cell culture dishes (1.0 × 10⁶ cells/cm²) for 72 h. The cell culture medium that contained the suspended cells was removed, and the adherent BMSCs were used for further analysis.

ORFV-GS14 preserved in our laboratory (an ORFV isolated from Gansu Province, China, designated as GS14) was used as a parent virus throughout the experiment and as a genetic background virus to generate target gene-deleted virus. Virus stocks were propagated in Primary Goat Testicular cells (PGT cells) and stored at −80 °C. The wild-type virus was prepared by sampling scabs from the goat and grinding them after the challenge experiment [3 (link)]. The cbp/gif double-gene deletion mutant (rGS14ΔCBPΔGIF) of ORFV was constructed in our laboratory [3 (link)]. The EGFP-N1 and other plasmids were kindly donated by the China Institute of Veterinary Drug Control. The PGT cells were prepared and frozen in our laboratory [3 (link),27 (link)]. Sheep testis cells were cultured in Ham’s DMEM/12 Gluta MAX medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS) (Every Green, Zhejiang Tianhang Biotechnology Co., PGTd, Hangzhou, China) and 1% penicillin-streptomycin solution (with a final concentration of 100 IU/mL of penicillin and 100 µg/mL of streptomycin) at 37 °C with 5% CO₂.