Hacat cell line

The human skin keratinocytes HaCat cell line and mouse epidermal JB6 Cl41 (JB6) cell line were purchased from ATCC, Virginia, USA. HaCat cells were cultured at 37°C in a 5% CO₂ incubator in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). JB6 cells were cultured at 37°C in a 5% CO₂ incubator in Eagle's minimum essential medium (MEM) supplemented with 5% FBS. Two kinds of cell lines were plated at 1×10⁵ cells per 6-cm dish and counted using a blood counting chamber.
Cells were seeded (8×10³ cells per well) in 96-well plates and cultured overnight, and then fed with fresh medium and treated with different concentration of cefradine. The cytotoxicity of cefradine was measured at different time points using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI). The absorbance was read at 490 nm according to the instructions.

Diethylene glycol single ethyl ether (Transcotol^® P) was obtained from Gattefossé (Lyon, France). Polyethylene glycol (PEG)-35 castor oil (Cremophor^® EL) was obtained from BASF (Ludwigshafen, Germany). Aconitine (purity> 98.0%) was supplied by Ze-lang BioScience (Nanjing, China). Methyl thiazolyltetrazolium (MTT) and materials used in cell culture were purchased from Gibco (ThermoFisher Scientific Inc., Grand Island, NY, USA). Hoechst 33,342 and LysoTracker Red were supplied by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Male Sprague–Dawley rats, weighing 300 ± 20 g, were provided by the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The study protocol was approved in April 2019 by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (license numbers: SYXK [Hu] 2008-0050, SYXK [Hu] 2009-0069).
The HaCaT cell line was obtained from ATCC (Manassas, VA, USA), and the CCC-ESF-1 cell line was provided by the Cell Culture Center of the Chinese Academy of Medical Sciences (Beijing, China).

Skin cancer A431, A375, A2058, SK-MEL-2 cell lines, and the normal skin HaCaT cell line were obtained from ATCC (Manassas, VA). Cells were grown in DMEM medium (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (FBS; Access Biologicals, Vista, CA) and 1% penicillin/streptomycin. Cell lines were maintained at 37 °C in a humidified cell culture incubator containing 5% CO₂. We also generated B-RAF^V600E mutant inhibitor vemurafenib resistance A375R and A2058R Cells. A375 and A2058 Cells were treated with 0.5–10 µM of vemurafenib. The cells were passaged twice every week to induce B-RAF^V600E mutant inhibitor resistance and drug resistance cells were maintained in 1 µM vemurafenib. All cell lines were grown for at least 24 h and used for experimentation when they reached 70–80% confluence.

Male Sprague–Dawley rats and nude mice weighing 200 ± 20 and 25 ± 5 g, respectively were used in this study, which was conducted with the approval of the Animal Ethical Committee of the Shanghai University of Traditional Chinese Medicine (permit SYXK [Hu] 2009-0069). All the animals were acclimatized at a temperature of 25 ± 2°C and relative humidity of 70 ± 5% for 1 week with food and water provided ad libitum. The HaCaT cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the CCC-ESF cell line was obtained from the Chinese Academy of Medical Sciences (Beijing, China).

The HaCaT cell line was obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C in a humidified incubator containing 5% CO₂. The cells were passaged at a ratio of 1:5 using 0.25% trypsin (Sigma-Aldrich, St. Louis, MO) and cultured to 70 to 80% confluence for the following experiments.

Human dermal fibroblasts were obtained from Clonetics (San Diego, CA, USA) and the human keratinocyte HaCaT cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Human dermal fibroblasts and the HaCaT keratinocyte cell line were cultured in Dulbecco’s modified Eagle’s media containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO₂ in air. Fibroblasts were plated at 90–95% confluence, and keratinocytes were plated at 70–80% in all experiments. The UV-B light source was provided. Keratinocytes and fibroblasts were pre-treated with 1 μg/mL RA, 5 mg/mL NAG, 5 μg/mL GPH, or 100–200 μg/mL fsCH and exposed to 30 mJ/cm² (keratinocytes) or 100 mJ/cm² (fibroblasts) UV-B radiation for 48 h.
The cytotoxicity of 1 μg/mL RA, 5 mg/mL NAG, 5 μg/mL GPH, and 100–200 μg/mL fsCH was determined after 24 h culture of keratinocytes and fibroblasts by using the MTT (3-(4,5- dimethylthiazol-yl)-diphenyl tetrazolium bromide, Duchefa Biochemie, Haarlem, The Netherlands) assay. Briefly, cells were maintained in a fresh medium including 1 mg/mL MTT at 37 °C for 3 h. Gentle shaking was conducted to dissolve purple formazan product in 0.5 mL isopropanol, and the absorbance of formazan was determined at λ = 570 nm using a Bio-Rad Model 550 microplate reader (Hercules, CA, USA).