Matrigel basement membrane matrix

Tumor formation by wild type (sAC FF) and Adcy10^−/− (sAC KO) melanocytes (n = 10 per cohort) was performed by injecting into both flanks 5,000,000 cells in 50 μL of 25% Matrigel Basement Membrane Matrix (Corning, #354248). Mice were observed every 3-4 days for tumor formation and tumor size was measured. Mice were euthanized when tumors reached 2 cm. For studies of the effects of cAMP microdomains on tumor growth, NSG mice were injected into the right flank with 5000 cells in 50 μL of 25% Matrigel Basement Membrane Matrix (Corning, #354248). Tumor growth was monitored every 3-4 days and the largest diameter was recorded. When tumors reached 1 cm in diameter, mice were randomized and one cohort was switched to doxycycline diet. When tumors in any cohort reached 2.5 cm in diameter, mice were euthanized, and tumor samples were collected for further analyses. Tumor growth data are presented as a fold change in size, normalized to tumor size on the day when mice were randomized and doxycycline was introduced into the diet. Upon euthanasia, tumors were removed, weighed, and samples were placed in formalin or flash frozen for subsequent analysis. Total number of mice per condition (regular chow/doxycycline chow): NLS-sAC 14/14, NES-sAC 9/9, mito-sAC 10/10, LacZ CTRL 5/5, 5SA-YAP 5/4, S397A-YAP 5/5, WT-YAP 5/5.

Primary cell lines derived from 6 proliferating IH samples from the 10 patients included in DAB IHC staining were sourced from the Gillies McIndoe Research Institute Tissue Bank, for analysis. These primary cell lines were cultured in Dulbecco’s Modified Eagle Medium (cat# 10569-010; Thermo Fisher Scientific, Waltham, MA) supplemented with 2% Corning Matrigel Basement Membrane Matrix (cat# 354234; Corning, NY), 10% fetal bovine serum (cat# 10091-148; Thermo Fisher Scientific), and 10,000 U penicillin–streptomycin (cat# 15140-122). The commercial cell line 3T3 (cat# CRL-1658; ATCC, Manassas, VA) was used as a positive control.

The invasive ability of cells was measured using the Corning^® Matrigel^® Basement Membrane Matrix (cat. no. 356234; Corning Inc., Corning, NY, USA) and a 24-well Transwell chamber (Corning Inc.) according to the manufacturer's protocol. The number of cells that passed through an 8-µm polycarbonate membrane was calculated. The polycarbonate surface of each chamber was covered with 50 µl Matrigel (1:8 dilution) to create an artificial basement membrane. Cells were cultured at 37°C in FBS-free MEM/F12 and RPMI-1640 medium for 24 h. After serum starvation, the cells were seeded at 1×10⁵ cells/well in the upper Transwell chamber, which contained ~200 µl serum-free medium. The lower chamber was filled with 500 µl of the medium supplemented with 10% FBS. After an incubation of 48 h at 37°C, the chambers were fixed at room temperature with paraformaldehyde for 30 min. Cells that had attached to the upper surface of the chambers were removed with a sterile cotton swab, and cells that adhered to the lower surface were stained with 0.1% crystal violet (Guangfu Institute of Superfine Chemical Industry, Tianjin, China) for 20 min at room temperature. The numbers of stained cells were counted using an inverted microscope (Olympus IX 70–142; Olympus Corp.) in 8 random fields. The experiment was repeated 3 times.