Dznep

Invasion assays were carried out by seeding 1 × 10⁵ cells on Corning BioCoat Tumor Invasion Systems (351158, Corning). The Corning FluoroBlok 24-Multiwell Insert System was used as a cell migration control (354166, Corning). Fetal bovine serum (10%) in RPMI 1640 was used as the chemoattractant. The invaded cells were labeled by Calcein AM fluorescent dye (354216, Corning) after 24-hour incubation at 37°C. Fluorescence of invaded cells was read at a wavelength of 494/517 nm (excitation/emission) (BioTek). The invasion percentage was calculated as {[mean relative fluorescence unit (RFU) of cells invaded through Corning Matrigel Matrix–coated membrane toward chemoattractant]/[mean RFU of cells migrated through uncoated Corning FluoroBlok membrane toward chemoattractant]} × 100. To examine the invasion of drug-treated melanoma cells, the cells were pretreated with DZNep (ApexBio Technology), EZP6438 (Selleck Chemicals), or GSK126 (Selleck Chemicals) for 3 to 6 days before seeding onto the upper chamber of the transwell system. Other experiments used a different BioCoat Tumor Invasion System (354165, Corning) and cell migration control (354578, Corning). For experiments using this system, noninvasive cells on the upper membrane of the insert were removed with Q-tips, and invasive cells attached to the bottom membrane of the insert were stained with crystal violet.