Dulbecco phosphate buffered saline (dpbs)

The MTT assay (3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (Sigma-Aldrich, Germany) was used to evaluate the effect of SETDB1 on cell viability (Na et al., 2016). MTT was dissolved in DPBS (GE Healthcare, USA). For the MTT assay, after transfection for 24 h, siRNA SETDB1 and the control cells were cultured with 100 µL of media in 96‐well plates (1–1.2 × 10⁴ cells/well) under standard conditions. The culture media were removed following incubation for 24 h, and the cells were washed with DPBS. Next, the MTT solution was added to the plate, which was kept for 4 h at 37 °C under standard incubation conditions. After incubation, the MTT solution was removed, and the formazan crystals on the plate were dissolved in 100 µL of dimethyl sulfoxide (Sigma-Aldrich, Germany). The absorbance at 570 nm was measured using a spectrophotometer (Shimadzu, UVmini-1240).

DMEM, MEM-α medium, 1 × DPBS, antibiotics, and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (Hyclone™, Logan, UT, USA). The EZ-Cytox cell viability assay kit was obtained from Daeil Lab Service Co., Ltd. (Seoul, Korea). Specific antibodies against phospho-Akt, phospho-cPLA₂, phospho-ERK, phospho-JNK, phospho-Lyn, phospho-p38, phospho-PKCδ, phospho-PLCγ1/2, phospho-Syk, and COX-2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A specific antibody against phospho-Fyn was obtained from Biorbyt Ltd. (Cambridge, UK). A specific antibody against β-actin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A specific antibody against phospho-5-LO and EIA kits for PGD₂ and LTC₄ were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). ELISA kits for IL-4 and TNF-α were purchased from e-Bioscience, Inc. (Science Center Drive, San Diego, CA, USA). A specific antibody against MMP-1 was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Specific antibodies against MMP-3 and MMP-13 were purchased from Abcam, Inc. (Cambridge, UK). 4-Nitrophenyl N-acetyl-β-d-glucosaminide (p-NAG), casein, DNP-HSA, DNP-IgE, trichloroacetic acid, caffeic acid, diethylene glycol, Folin-Ciocalteu reagent, and quercetin were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.

MEM-α medium, 1 × DPBS, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (Hyclone™, Logan, UT, USA). The EZ-Cytox cell viability assay kit was obtained from Daeillab Service Co. (Seoul, Korea). Specific antibodies against phospho-cPLA₂, phospho-ERK, phospho-JNK, phospho-LYN, phospho-p38, phospho-PKCδ, phospho-PLCγ1, phospho-SYK and COX-2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A specific antibody against phospho-FYN was obtained from Biorbyt Ltd. (Cambridge, UK). A specific antibody against α-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). An enzyme immunoassay (EIA) kit for PGD₂ was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, and IL-6 were purchased from e-Bioscience, Inc. (San Diego, CA, USA). 4-Nitrophenyl-N-acetyl-β-d-glucosaminide (p-NAG), dinitrophenyl-human serum albumin (DNP-HSA) and DNP-immunoglobulin E (DNP-IgE) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.

To assess metabolism of cells cultured on the surface of the membranes, we used a resazurin cell metabolism assay. In this assay, resazurin (an oxidised non-fluorescent blue colored compound) is reduced by metabolically active cells to resorufin (a fluorescent pink product), which is detected via fluorescence measurement. Resazurin solution (5 mg resazurin salt in 40 mL DPBS) (PAA laboratories), was added to each sample (100 μl/ml) and returned to the incubator for 4 h. A 100 μl aliquot of media was taken from each sample and transferred into a black bottomed 96-well plate. Fluorescence was measured at 530–510 nm excitation and 590 nm emission using a fluorescence reader (FLUOstar OPTIMA, BMG LABTECH) (n = 4).

Hypoxia response was evoked by exposure to reduced oxygen levels (1% O₂) in a hypoxia chamber (Sci-tive-N hypoxia workstation, Ruskinn Technology, Bridgend, UK) and control cells were cultured in atmospheric oxygen. Samples were taken after 48 and 72 hours. The hypoxia chamber was manually calibrated, and oxygen levels were verified frequently. Following hypoxia exposure, cells were kept on ice, rinsed with cold D-PBS (PAA Laboratories) and lysed directly in the culture flask to reduce HIF1Α protein degradation. Complementary experiments with CoCl₂ (Sigma-Aldrich, St Louis, MO) were performed to validate the hypoxia data. CoCl₂ treatment (150 µM) was carried out under normal atmospheric oxygen for 48 or 72 hours.