Hacat

The HaCaT (human keratinocyte) cell line was purchased from the Korean cell line Bank (Seoul, Republic of Korea) and the RAW 264.7 (mouse macrophage) cell line was purchased from the American Type Culture Collection (ATCC, VA, USA) (ATCC; TIB-71). The cell lines were maintained in DMEM containing 10% heat-inactivated FBS and 1% of penicillin (100 units/mL)/streptomycin (100 μg/mL) (Welgene, Seoul, Republic of Korea). The cell lines were cultured in an incubator with a humidified atmosphere of 5% CO₂ at 37 °C, and the medium was changed every 2 or 3 days during incubation.

Human cancer cell lines (colon cancer cell lines: SNU-81, SNU-C4, and HCT-116; breast cancer cell line: MDA-MB-231; gastric cancer cell lines: MKN-1 and SNU-484; glioma cell line: C-6) and normal cell lines (HEK293 and HaCaT) were obtained from the Korean Cell Line Bank (KCLB) (Seoul, Korea). 293FT cells were purchased from Life Technologies (Carlsbad, CA, USA).

The standard reference materials (SRM) 1649b were purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA). PM particles at concentrations of 25 μg/cm² were prepared in serum-free medium.
Interleukin-1 alpha (IL-1α) and polyinosinic:polycytidylic acid sodium salt (Poly I:C), used to produce the AD-like cell model, were obtained from PeproTech (Cranbury, NJ, USA) and Sigma-Aldrich (St Louis, MO, USA). For western blotting, an anti-IL-4 antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). An anti-IL-6 antibody was purchased from Cell Signaling (Danvers, MA, USA). An anti-IL-1β antibody was purchased from MyBioSource (San Diego, CA, USA). An anti-GAPDH antibody was obtained from Santa Cruz (Delaware Avenue, CA, USA). An anti-FLG antibody was purchased from Life Span Biosciences (Seattle, WA, USA). Anti-rabbit IgG, anti-mouse IgG, and HRP-linked antibodies were purchased from Cell Signaling Technology. The human immortalized keratinocytes (HaCaT) and human dermal fibroblast (HDF) cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). The human mast cell line HMC-1 was obtained from Merck Millipore (Temecula, CA, USA).

The human keratinocyte cell line, HaCaT, was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

Human mast cell line (HMC-1), human keratinocyte cell line (HaCaT), and murine macrophages cell line (RAW 267.4) were purchased from the Korean Cell Line Bank (Korea). HMC-1 were cultivated in Iscovés Modified Dulbeccós Medium (Lonza, Switzerland) added with 10% MSC-qualified fetal bovine serum (FBS)(Gibco BRL, USA), HaCaT in Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza) containing 10% FBS, and RAW 264.7 cells in DMEM supplemented with 10% FBS. All three cultures were kept at 37°C in 5% CO₂ incubator.

The spontaneously immortalized human keratinocyte cell line HaCaT was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 unit/mL penicillin and 100 μg/mL streptomycin. Cells were then incubated in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were treated with RR for 1 h and stimulated with or without TNF-α and IFN-γ (each 10 ng/mL) for 24 h in serum-free culture medium. The KeraSkin™ model (Biosolution, Seoul, Korea) was transferred onto a 6-well plate with 1 mL of KeraSkin growth medium and incubated overnight at 37 °C with 5% CO₂. To induce skin inflammation, TNF-α or IFN-γ (10 ng/mL), dissolved in a solution of polyethylene glycol and PBS (1:1), was applied twice a week before sample treatment. TNF-α or IFN-γ was removed with repeated rinsing with PBS, and Dex (1% (w/v)), NR (1% (w/v)), RR (1% (w/v)), and RSV (1% (w/v)) were applied thrice weekly for two weeks.

HaCaT (Immortal human keratinocyte) cell line was purchased from Korean Cell Line Bank (Seoul, Republic of Korea) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; WELGENE Inc., Daegu, Republic of Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning, NY, USA) and 1% penicillin–streptomycin (Gibco, New York, NY, USA) in a humidified incubator at 37 °C and 5% CO₂.