MAPK and Notch-mediated effects of Meso-Xanthin F199 compounds on proliferative activity and apoptosis of human melanocytes in three-dimensional culture.
The complete proteome shared between assayed melanocytes cultured in monolayer (M2319) and in spheroids before (Nm01) and after (Am02) exposure to Meso-Xanthin F199™. Data content comprises of assigned genes attribution, proteins characterization according to the obtained proteomic analyzes, and table of the semi-quantitative distribution of the defined proteins (green color indicates the most abundant; red color indicates the least abundant proteins among analyzed cell cultures; semi-quantitative data are estimated in linear and logarithmic scales).
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Cultivation of melanocytes The study was performed using the primary culture of human melanocytes (104-05N, CELL Applications, Inc). Upon transportation cells were thawed, resuspended in a complete melanocyte growth medium provided by the manufacturer (135-500, CELL Applications, Inc) and placed on culture dishes at a density of 104 cells/cm2 (monolayer cells designated as M2319). Full growth medium was replaced every two-three days. The culture was passaged upon reaching a 60% confluent state. For the further proteome studies, suspension of melanocytes at passage 4 were used to obtain spheroids under 3D non-adhesive conditions in agarose plates as described previously (14). Meso-Xanthin was added to the growth medium of experimental spheroids (cells designated as Am02) at 1:10 ratio (normalized to 50 M of fucoxanthin as the main compound of Meso-Xanthin™ F199). The same volume of sodium chloride (NaCl) solution was added to the growth medium in the control group (cells designated as Nm01). Spheroids from experimental and control groups were harvested and collected on day 7 in 1.5mL tubes, and the growth medium was completely removed by centrifugation in phosphate buffer saline. The resulting pellets of only spheroid cells were frozen at -80°C and stored until samples preparation for proteomic analysis. Liquide chromatography and Mass spectrometry Liquid chromatography was carried out on an Ultimate 3000 RSLC Nano (Thermo Scientific) system. Peptides were loaded onto an enrichment column Acclaim Pepmap® (5 mm x 0.3 mm, 300A pore size, 5 m particle size) for 6 minutes at a flow rate of 20 L/min in water with 3.5% acetonitrile supplemented with 0.1% formic acid and 0.05% acetic acid (mobile phase C, pH=2.75±0.1 at 20±2oC). After enrichment, peptides were washed out and separated for 90 minutes (including column equilibration time 10 minutes) onto an analytical column Acclaim Pepmap® (75 m х 150 mm, 1.8 m particle size, 60А pore size) at 0.30 L/min flow rate in a gradient of mobile phase A (water; pH 2.6±0.15 at t = 20±2oC) and mobile phase B (70% acetonitrile and 30% methanol) both supplied with 0.1% formic acid and 0.03% acetic acid. Detection of peptides signal was accomplished using a high-resolution benchtop Orbitrap Q-Exactive HF-X (Thermo Scientific, USA) mass spectrometer operated in a positive ionization mode and equipped by a nano-flow NSI ions source.