Ez cytox reagent

Phosphorylation-specific antibodies against JNK, ERK1/2, p38, IκBα, and p65, and antibodies against JNK, ERK1/2, p38, IκBα, p65, iNOS, glyceraldehyde 3-phosphate dehydrogenase, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse interleukin-6 (IL-6) and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from BD Biosciences (San Jose, CA, USA). EZ-Cytox reagent was purchased from DoGen Bio (Seoul, Republic of Korea). Sulfanilamide, phosphoric acid, and naphthyl ethylenediamine dihydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lipopolysaccharides from E. coli 0111:B4 (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum were purchased from Gibco (Grand Island, NY, USA). RPMI 1640 medium came from Cytiva (Marlborough, MA, USA), and penicillin/streptomycin antibiotics came from Invitrogen (Carlsbad, CA, USA). EZ-Cytox reagent was obtained from DoGenBio (Seoul, South Korea). Griess reagent, dimethyl sulfoxide (DMSO), protocatechuic acid, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ELISA kits for PGE₂, TNF-α, IL-6, IL-8, and IL-1β were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Isookanin was obtained from ChemFaces Biochemical Co. Ltd. (Wuhan, Hubei, China), and dissolved in DMSO. The luciferase assay system and lipofectamine™ 3000 reagent were purchased from Promega Co. (Madison, TN, USA) and Invitrogen (Waltham, MA, USA), respectively. Mitogen-activated protein kinases (MAPKs) antibodies (p-ERK1/2, ERK1/2, p-p38, p38, p-SAPK/JNK, SAPK/JNK) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), while secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

A375 and A375-VR cells (1000 cells/well) and Hs294T cells (2000 cells/well) were seeded in 96-well plates. The next day, inhibitors and vemurafenib were treated at indicated concentrations. The WST assay was performed using EZ-cytox reagent (DogenBio) diluted in RPMI1640. The absorbance at the 450-nm wavelength was measured using Wallac EnVision (Perkin Elmer) 3 days after drug treatment. Drug synergies were evaluated by performing SynergyFinder (https://synergyfinder.org) [82 (link)].

Iscove’s modified Dulbecco’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/streptomycin antibiotics (Invitrogen, Carlsbad, CA, USA) was used as culture media for culturing HMC-1 in a humidified incubator at a temperature of 37 °C and CO₂ level of 5%. The cells (5 × 10⁵ cells/mL) were seeded in sterile 6-well dishes for 16 h and treated with or without Apigenin at indicated concentrations for 1 h, after which the cells were stimulated with 50 nM of phorbol-12-myristate 13-acetate and 1 µM of calcium ionophore A23187 (PI) for indicated times. Apigenin with purity of 98% (ChemFaces, Wuhan, China) was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and stored at −20 °C before application. The final concentration of dimethyl sulfoxide in the cell culture media was below the toxicity level of p < 0.01%.
For cell viability assay, WST assay was employed to determine cell viability. Cells were pre-treated with various concentrations of Apigenin (0, 5, 10, 20, and 30 μM) for 24 h and 0.01 mL of EZ-Cytox reagent (Dogenbio, Seoul, Korea) was added before further incubation for 4 h. After incubation, the absorbance was measured at 540 nm with a microplate reader (Tecan, Männedorf, Switzerland). The absorbance correlates with cell viability.

HaCaT and HMC-1 cell viability was measured using the EZ-Cytox reagent (EZ-1000, Dogenbio). The HaCaT cells (1 × 10⁴ cells/cm²) and HMC-1 (1 × 10⁵ cells/mL) were seeded in a 96-well plate for 24 h. For the HMC-1 cells, due to the characteristics of the suspension cells, the treatment of the samples was started immediately 30 min after seeding. The cells were then pretreated with different concentrations (0.05–1%, concentrations of extracts show diluted content based on 10 brix of EEA) of EEA for 24 and 48 h. After incubation, 0.01 mL EZ-Cytox reagent was added to each well and incubated for 2 h. The absorbance in each well was then measured at 450 nm using a microplate reader.