H ecm liposome

KCs (2 × 10⁴ cells/well) and HDF cells (2 × 10⁴ cells/well) were seeded into 12-well plates and treated with 0.05% H.ECM™ liposome or positive control (1 μM retinoic acid (Sigma-Aldrich) or 10 ng/mL recombinant epidermal growth factor (EGF; R&D systems, Minneapolis, MN, USA) when the cells reached around 80% confluence. Then, the UVB-irradiated human skin specimens were homogenized using TissueLyser Ⅱ (Qiagen, Hilden, Germany). Total RNA was extracted using the RNAiso Plus (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA was quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher) and reverse transcribed using an RNA to cDNA EcoDry Premix Kit (Takara Sake, Berkley, CA, USA). The synthesized cDNA, Taqman Gene Expression Master Mix (Applied Biosystems), and Taqman primer of each target (HAS3; Hs00193436_m1, AQP3; Hs00185020_m1, COL1A1: Hs00164004_m1, MMP1: Hs00899658_m1; Filaggrin; Hs00856927_g1, Loricrin; Hs01894962_s1, Applied Biosystems) were used for the qRT-PCR experiments. The expression level of each gene was normalized to that of the housekeeping gene GAPDH (Hs02786624_g1, Applied Biosystems) and calculated using the 2^−ΔΔCt method.

RAW 264.7 cells (2 × 10⁴ cells/well) were seeded in 12-well plates and treated with LPS (2 μg/mL; Sigma-Aldrich, Saint Louis, MO, USA) for inducing TNF-α expression when cells reached more than 80% confluence. The cells were treated 0.1% H.ECM™ liposome or 1 μM Dexamethasone (positive control; Sigma-Aldrich) and incubated for 24 h. Then, the media used at culture was centrifuged at 2000× g for 10 min, and the supernatant was used for ELISA. The Mouse TNF-α ELISA kit (Abcam, Cambridge, MA, USA) was used for TNF-α measurement. ELISA was performed according to the manufacturer’s instructions. The absorbance at 450 nm was measured using an ELISA microplate reader (VersaMax; Molecular Devices, California, CA, USA).