Tpca 1

Human coronary artery endothelial cells (HCAECs) were purchased from Procell (Wuhan, China). The cells were cultured in a complete culture medium for HCAECs (Procell) at 37°C, 95% air, and 5% CO₂ conditions.
To establish an IVIG resistance system, HCAECs were seeded into six-well plates and cultured until they reached 90% confluence. The cells were then incubated with RPMI-1640 medium supplemented with 5, 10, or 15% serum from patients with IVIG-resistance, IVIG responses, and healthy controls for 48 h. In addition, HCAECs were incubated with 15% serum for 0, 24, 48, and 72 h to screen induction time.
To inactivate the NF-κB pathway, 1.0 mmol/L TPCA-1 (IKK-2-specific inhibitor; MedChem Express, Monmouth Junction, USA) was added and incubated with HCAECs for 24 h.

Canine melanoma cells (MCM-N1 cell line; 13-year-old male dog; chromosome number, 2n = 74) obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). The other canine melanoma cell lines (CMe1, CMMe2 and LMe) were kindly provided by Dr. Takayuki Nakagawa (Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo) [39 (link)–41 (link)]. The cells were cultured in 75-cm² culture flasks containing DMEM-LG (FUJIFILM Wako Chemical Corp., Osaka, Japan) supplemented with 10% FBS, 100 unit/mL penicillin and 100 μg/mL streptomycin, and maintained at 37°C in the environment of a humidified incubator with 5% CO₂ as described previously [42 (link), 43 (link)]. The cells were treated with recombinant canine IL-1β (Kingfisher Biotech, Inc., Saint Paul, MN, USA) for the indicated periods and concentrations. For inhibitor treatment, the cells were pretreated with 2-((aminocarbonyl)amino)-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1, 10 μM for 1 h, MedChemExpress, Monmouth Junction, NJ) or UK356618 (50 nM for 2 h, MedChemExpress, Monmouth Junction, NJ), then the cells were treated with recombinant canine IL-1β.

We cultured A549 cell lines in McCoy’s 5A (GE Healthcare Life Sciences, Freiburg, Germany) plus 10% fetal calf serum (FCS; Thermo Fisher Scientific, Darmstadt, Germany) at 37°C. The medium contained penicillin and streptomycin. For A549 p53-Venus reporter cells [64 (link),65 (link)], selective antibiotics (400 μg/ml G418 (Carl Roth, Karlsruhe, Germany) and 50 μg/ml hygromycin (Thermo Fisher Scientific)) were added to maintain transgene expression.
For corresponding treatment of the cells, the medium was replaced with fresh one containing dimethyl sulfoxide (DMSO; Sigma-Aldrich) as a control, IKK2 inhibitor (15 μM TPCA-1, 120 μM sc-514 or 0.93 μM BMS-345541 (MedChemExpress, Sollentuna, Sweden)), 10 ng/ml or 50 ng/ml IL-6 (PeproTech, Hamburg, Germany) or 10 ng/ml TNFα (Enzo Life Science, Lörrach, Germany) 1 h before irradiating cells (if not stated otherwise) with X-rays at a dose rate of 1 Gy/26 s (250 keV, 10 mA).