MC1R Receptor Binding with [125I]-Nle4-D-Phe7-α-MSH.

MAPK and HDAC activity were inhibited by BRAF_i (1−10 μM dabrafenib) and HDAC_i (0.5−10 mM 4-phenylbutyrate), respectively, in A2058 cells. For MEWO cells, HDAC activity was inhibited by incubation with 4-phenylbutyrate (0.5−10 mM) and vorinostat (0.5−10 μM). Treatments were carried out for 12−24 h in flat-bottom polystryene 24-well plates. MC1R-radioligand binding assays were conducted using synthetic-radiolabeled α-MSH analogue [¹²⁵I]-Nle⁴-D-Phe⁷-α-MSH ([¹²⁵I]-NDP-α-MSH, NEX352010UC, PerkinElmer). Briefly, cell culture media containing the drugs were aspirated, followed by a gentle wash with 0.5 mL of binding media (modified Eagle’s medium with 25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid, 0.2% BSA, and 0.3 mM 1,10-phenanthroline). Binding media (0.3 mL) containing ~20 000 cpm [¹²⁵I]-NDP-α-MSH were added to cells and incubated for 2 h at 25 °C. Following the radioligand binding, the cells were rinsed gently twice with0.5 mL of ice-cold 10 mM PBS buffer with 0.2% BSA and lysed in 0.5 mL of 0.5 M NaOH for 5 min. Cell lyses were harvested, and radioactivity was measured using standard gamma counting (Packard Cobra 5002, PerkinElmer).