Figure 5
Estrogen exposure influences leptin expression in ASCs. (A) Adipose stem cells ASCs (n = 6 donors per group) were cultured in media supplemented with charcoal dextran stripped FBS (CDS-FBS) supplemented with (E2) or ICI182,780. Cells were collected at 70% confluency and total RNA was isolated for real-time PCR analysis of leptin expression. (B) Cell lysate was collected from n = 6 ASC donors cultured in CDS-FBS with or without E2 at 70% confluency and subjected to Western blot analysis with 20 μg of protein and separated by SDS-page under reducing conditions, blotted and probed with antibodies to leptin or actin. (C) Cell lysate was collected from six different ASCs donors and either pooled or analyzed as individual donors (D#), cultured in CDS-FBS with or without estrogen. A total of 20 μg of protein was separated by SDS-page under reducing conditions, blotted, and probed with leptin and actin antibodies. (D) ASCs (n = 6 donors per group) were co-cultured with MCF7 cells and treated with vehicle (dimethyl sulfoxide, DMSO) neutralizing leptin antibody (Lep nAb), estrogen (E2) or neutralizing leptin antibody and estrogen (Lep nAb + E2) in complete culture media (CCM) containing CDS-FBS. After seven days, the number of MCF7 cells was calculated based on the percentage of GFP+ MCF7 cells by the total number of cells in the population. (E) ASCs (n = 6 donors per group) were cultured in CCM and collected at 70% confluency and total RNA was isolated for real-time PCR analysis of aromatase expression. (F) ASCs (n = 6 donors per group) were co-cultured with MCF7 cells and treated with letrozole (10 nM) in CCM. After seven days, the number of MCF7 cells was determined by the percentage of GFP+ cells multipled by the total number of cells. Percent change was determined by the relative change in the number of MCF7 cells after letrozole treatment compared to their non-treated controls. Values reported are the mean of three independent experiments, each performed in triplicate. Bars, ± SD. *, P <0.05; **, P <0.01; #, P <0.005.