High glucose dulbecco s modified eagle s medium

The human immortalized keratinocyte cell line, HaCaT, was purchased from Addex Bio Technologies (AddexBio Technologies, San Diego, CA, USA). Cells were equilibrated in a 5% CO₂ incubator at 37 °C and grown in high-glucose Dulbecco’s Modified Eagle’s Medium (HyClone Laboratories Inc, Grand Island, NY, USA). At 80% confluency, cells were starved in serum-free medium for 24 h, pretreated with 25, 50, or 100 µg/mL ADE for 1 h, and induced with 10 ng/mL TNF-α and 50 ng/mL IL-17a (Peprotech, Rocky Hill, NJ, USA) for 23 h before RNA extraction or for 30 min before protein extraction. Cells were incubated with wortmannin, a specific Akt inhibitor, and AG490, a JAK2 inhibitor, for 4 h before being stimulated for 20 h with a combination of IL-17a/TNF-α.

Human ovarian cancer cell lines SKOV3 and OVCAR3, and breast cancer cell lines Hs578t and MDA‐MB‐231 were purchased from the Tumor Cell Bank of the Chinese Academy of Medical Sciences (Shanghai, China). OVCAR5, OVCAR8 and OVISE were provided by Tongji Medical College, Huazhong University of Science and Technology. All cell lines were confirmed by short tandem repeat DNA fingerprinting. The cells were cultured in a humidified incubator with 5% (v/v) CO₂ at 37 °C. Hs578t and MDA‐MB‐231 were cultured in high‐glucose Dulbecco's modified Eagle's medium (HyClone Laboratories, Logan, UT, USA). Other cells were cultured in RPMI‐1640 medium (HyClone Laboratories) with 10% fetal bovine serum (FBS; Biosera, Shanghai, China) and 1% penicillin/streptomycin (Beyotime Institute of Biotechnology, Shanghai, China).

Adipose-derived MSCs (ADMSCs) were obtained from adipose tissue from three healthy human donors as described previously (Ganbold et al., 2019 (link)). All experimental procedures in this study were approved by the Institutional Review Board of the School of Dentistry, Seoul National University (IRB No. S-D20150019). In brief, lipid tissue was collected from liposuction specimens, digested with 0.1% collagenase I (Gibco, Carlsbad, CA) in Hanks’ balanced salt solution (HyClone Laboratories, Logan, UT), and passed through a 100-µm strainer (BD Falcon, Franklin Lakes, NJ). Cells were resuspended in high-glucose Dulbecco’s modified Eagle’s medium (HyClone Laboratories) containing 10% fetal bovine serum (HyClone Laboratories). All cultures were maintained in a humidified incubator at 37°C and 5% CO₂. Cells were passaged at ∼70% confluence, and cells at passages 3 to 7 were used. To generate MiBs, ADMSCs were seeded on AggreWell™400 plates (Stem Cell Technology, Cambridge, MA) and cultured for the 1, 2, 3, or 7 days to form MSC spheroids. To collect spheroids of uniform size, spheroids were passed through double-stacked cell strainers with pore sizes of 70 μm and 300 μm. MiBs were imaged using the JuLi stage system (NanoEnTek, Seoul, Korea), and MiB diameter was measured using ImageJ software (National Institutes of Health, version 1.53j, https://imagej.nih.gov/ij/download.html).