Raw264.7 macrophage

Human cervical squamous carcinoma cell lines (SiHa and C33a) and murine RAW264.7 macrophages were purchased from ATCC and cultured according to their guidelines_. Human monocytic cell line THP-1 was obtained from the China Center for Type Culture Collection (CCTCC) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Hypoxia treatment was administered by placing the cells in a hypoxic incubator (Thermo Scientific, USA) maintained at a low oxygen concentration (1% O₂, 5% CO₂ and 94% N₂).

The RAW 264.7 macrophages (ATCC, Manassas VA) were cultured in DMEM medium supplemented with 10% FBS (Life Technologies) and 1% penicillin-streptomycin (GE Healthcare Life Sciences) and maintained in a humidified incubator at 37°C and 5% CO₂.

A549 lung cells (ATCC, Manassas, VA, USA; CCL-185), 293 T embryonic kidney cells (ATCC; CRL-3216), RAW264.7 macrophages (ATCC; TIB-71) and Calu-3 lung cells (Hunan Fenghui Biotechnology Co., Ltd, Hunan, PRC; CL0062) were maintained in complete medium of Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS), and 1% antibiotic–antimycotic (Invitrogen, Waltham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37 °C. Cell subculture was performed at 60–70% confluence.

RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium High Glucose (4.5 g/L) and NaHCO₃ (1.5 g/L) (DHG-L1, Gibco-Life Technologies, Carlsbad, CA, USA), supplemented with 10 % heat-inactivated foetal bovine serum (FBS, Gibco). HeLa cells (ATCC) were cultured in Minimum Essential Medium (MEM, Gibco) supplemented with nonessential amino acids (Gibco), 1 mM pyruvate (Gibco) and 10% FBS. DRP1^+/+ and DRP1^−/− MEFs (a generous gift from Prof. Ishihara, Kurume University, Japan) were kept in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10 % FBS and EB1 and rho⁰ HeLa cells (kind gift from Prof. Hayashi, University of Tsukuba, Japan) in DMEM high glucose (DHG, Gibco) supplemented with 1 mM pyruvate, 50 µg/ml uridine (Sigma-Aldrich, St. Louis, MO, USA) and 10% FBS. BMDMs were obtained from femurs and tibias of 6-to-8-week-old C57BL/6 mice, as previously described^{94 (link)}. Cells were seeded and treated in culture plates (Corning-Costar, Lowell, MA, USA).
When indicated, myxothiazol (10 nM, Sigma-Aldrich), antimycin A (100 nM, Sigma-Aldrich), Mito-TEMPO (500 µM, Sigma-Aldrich), ruthenium red (1 or 10 µM, Sigma-Aldrich), tunicamycin (10 µM, Sigma-Aldrich), TNFα (10 ng/ml, Sigma-Aldrich) or cycloheximide (10 µg/ml, Sigma-Aldrich) were added to the culture media for the indicated times.

RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were maintained in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% (v/v) FBS (Gibco, Waltham, MA, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin, and incubated at 37 °C and an atmosphere of 5% CO2. Cells were serum-starved overnight in DMEM with 1% FBS and 100 μg/mL streptomycin, and 100 U/mL penicillin before being treated with 5 mM D-glucose (LG) DMEM, 25 mM D-glucose (HG), for 4, 8, 24 h according to [42 (link)]. The pharmacological inhibitors specific to PI3K, LY294002, were purchased from Selleckchem and were applied at 10 uM for 30 min before glucose treatment. RAW cells in this study were analyzed at passage 2 to 3. Wound macrophages will be harvested as described [43 (link)], 10⁵ wound macrophages can be routinely obtained per 100 mg wounds, and we have confirmed that these cells are ~98% macrophages based on F4/80 and CD11b staining.

Cryopreserved RAW264.7 macrophages purchased from ATCC (Manassas, VA) were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum (FBS). The cells were cultured at 37°C, and the medium was changed every 3 days when the cell confluence was approximately 80%.
The RAW264.7 cells were seeded into 24-well plates at 1 × 10⁵ cells/well, and divided into 6 groups: Control group, LPS group, 2D-sEVs group, 4D-sEVs group, IGFBP2-siRNA-#2 and NSC74859 group. First, the LPS group, 2D-sEVs group, 4D-sEVs group, IGFBP2-siRNA-#2 and NSC74859 group were stimulated with 100 ng/mL lipo-polysaccharide (LPS, Sigma-Aldrich, Waltham, MA, United States) for 12 h, and the Control group contained an equal volume of medium without LPS. Next, the supernatant was removed and washed 3 times with PBS. STAT3 inhibitor, S3I-201(NSC74859, S1155; Shanghai, China) is used as described in the article (Zheng et al., 2019 (link)). NSC74859 group was pre-treated with S3I-201 (10 µM) for 1 h. Dil dye-labeled 2D sEVs (40 μg/well) were added to the 2D-sEVs group, and an equal amount of Dil-labeled 4D sEVs/IGFBP2-siRNA-#2-4D sEVs were added to the 4D-sEVs/NSC74859/IGFBP2-siRNA-#2 group for 24 h. Dil dye was added to the Control and LPS group for 24 h. The superna-tant was collected, and the cells were fixed for immunohistochemical staining.

Human hepatoma HepG2 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM, 5.5 mM glucose) containing 10% fetal bovine serum (FBS) and antibiotics (1% penicillin-streptomycin) at 37°C in 5% CO₂ in air. Mouse AML-12 liver cells (ATCC, Manassas, VA, USA) were grown and maintained in 1 : 1 mixture of DMEM and Ham's F12 medium with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, 40 ng·mL dexamethasone, 10% FBS, and antibiotics (1% penicillin-streptomycin) at 37°C in 5% CO₂ in air. Mouse RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were grown and maintained in DMEM containing 10% fetal bovine serum (FBS) and antibiotics (1% penicillin-streptomycin) at 37°C in 5% CO₂ in air. Media were replaced with fresh medium every 2 to 3 days. Cells were split at a 1 : 4 ratio at 70 to 80% confluence.
Lipid accumulation in HepG2 cells was induced by incubation in 25 mM glucose DMEM media for 48 hours. Lipid accumulation and inflammatory response in AML-12 cells and RAW 264.7 macrophages were induced by stimulation with 500 μM free fatty acids (FFA, palmitic-oleic acid mixture 1 : 2) and lipopolysaccharide (LPS, 1 ng/mL) for 24 hours.
Treatment (metformin 0.1 mM, leucine 0.5 mM, and sildenafil 1 nM) was added for further 24 to 48 hours.