Leptin Signaling in Myometrium and Leiomyoma

The primary myometrium and leiomyoma cells were treated with 100 ng/mL leptin for 48 h. Human adipocyte (SW872) cells were used to coculture with human primary or immortalized myometrium and leiomyoma cells [10 (link)]. We used a Transwell system using 0.4-mm porous membranes from Corning (Corning, New York, NY, USA) to coculture the cells with their respective media. We cultured the Transwell system for eight days, and changed 50% of the medium every 48 h. We then collected the myometrium and leiomyoma media for the multiplex array, and for protein quantification and analysis, we harvested the cells. For a separate series of experiments, the cultured single cells and adipocyte cocultured cells for 7 days were treated with the STAT3 inhibitor (AG490, Calbiochem, Merck Millipore, Burlington, MA, USA) at 50 μM, ERK the inhibitor (PD98059, Cell signaling, Danvers, MA, USA) at 10 μM, and the AKT inhibitor (MK2206, Cayman Chemicals, Ann Arbor, MI, USA) at 10 μM for 24 h. For the leptin treatment, we treated the cells with 100 ng/mL leptin for the first 24 h and then the combination with inhibitors for another 24 h. After 24 h of incubation, we harvested the cells for protein quantification and analysis.