Sch772984

Trabectedin was gifted by TAIHO Pharmaceutical, Co. LTD (Tsukuba, Japan). Methotrexate (MTX) hydrate and doxorubicin (DOX) were purchased by Sigma‐Aldrich (St. Louis, MO, USA) and Wako Pure Chemical Industries (Osaka, Japan), respectively. The ERK inhibitor, SCH772984, was purchased from Cayman Chemical (Ann Arbor, MI, USA). The drugs were prepared in dimethyl sulfoxide (DMSO) and stored at −20°C. For in vitro examinations, the drugs were diluted in DMEM to the desired concentration. For in vivo experiments, trabectedin was further diluted with phosphate buffer pH 4.0 immediately before administration.
The following primary antibodies were used: anti‐cleaved caspase‐3 and anti‐beta‐actin (Cell Signaling Technology, Inc., Danvers, MA, USA); anti‐microphthalmia‐associated transcription factor (MITF) and anti‐activating transcription factor 1 (ATF1) (Abcam, Cambridge, UK); anti‐PCNA, anti‐tyrosinase (TYR), and anti‐tyrosinase‐related protein 2 (TRP2) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Horseradish peroxidase (HRP)‐conjugated secondary antibody was obtained from GE Healthcare Life Sciences (Pittsburg, PA, USA).

Cells were seeded in 12 mm Transwell^® inserts as indicated in the ACRE Model Setup. On day 4 of the ACRE Model Setup, prior to exposure, seeded ACRE inserts were transferred to a temporary multi-well plate. Pre-warmed (37 ˚C) basal exposure medium containing the ERK1/2 and p38 MAPK inhibitors, 800 nM SCH772984 (Cayman Chemical, #19166) and 300 nM LY2228820 (mesylate) (Cayman Chemical, #23259), respectively, was added to the apical and basolateral compartments of the multi-well plate. ACRE inserts were returned to the tissue culture incubator for 2.5 h. Following incubation, basal exposure medium containing MAPK inhibitors was removed, and ACRE inserts were combined with seeded HULEC and exposed as described in ACRE Model Setup. Western blot analysis was performed on H441 cellular protein extracts after 1 h VEH and 1 h ACRE-DEP treatment and qPCR analysis was performed on synthesized HULEC cDNA after 6 h VEH and 6 h ACRE-DEP treatment. This methodology was then repeated with an additional set of ERK1/2 and p38 inhibitors, 1 µM BVD523 and 3 µM SB203580, respectively. Statistical analysis was conducted in GraphPad Prism (version 9.3.1) using an ordinary one-way ANOVA and Šidák’s multiple comparisons post-hoc test.