Coculture analysis was performed in 6- or 12-well Millicell Hanging cell culture inserts (1.0 μm of polyethylene terephthalate; MCRP06, MCRP12; Millipore). Pre-adipocytes were either seeded (coculture) or not seeded (monoculture; control) into hanging inserts and induced to differentiation for 9 to 11 days. At differentiation, WM3682 cells were seeded into the lower compartment of the transwell, and the cells were cocultured for 5 days in nutrition medium. For reverse coculture analysis, the hanging inserts, containing adipocytes, were removed after 5 days of coculture, medium was replaced to unconditioned medium, and cells were allowed to grow for an additional 5 days. Cells were trypsinized to maintain 70% confluency. For assays with adipocyte-conditioned medium, medium from culture of differentiated adipocytes was replaced 8 days after differentiation induction, and after an additional 2 to 3 days, supernatants were collected on ice. Cells and cellular debris were excluded from the medium by centrifugation at 12,000 rpm for 5 min.

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