Western blot MCF7 cells and FACS sorted MCF7 cells immediately after co cultured with ASC donors have been incu bated in phosphatase and protease inhibitors, lysed with RIPA buffer, and centrifuged. Cell lysate was also obtained from Ob Ab, Ob Ab, Ob Ab, or Ob Ab ASCs cultured in CCM created with charcoal dextrose stripped FBS, Exactly where indicated, ASCs were treated with 10 nM 17B estradiol and or one hundred nM ICI182,780, and cell lysate was obtained. A total of 20 ug of protein was fractionated on four to 12% SDS polyacrylamide gels and transferred to nitrocellulose mem branes, The blots have been blocked with bl k Noise Canceling Reagents and probed making use of primary antibodies incubated overnight at four C, washed with phosphate buffered solu tion with 0.
01% TWEEN selleck inhibitor 20, followed by a sec ondary antibody conjugated to horseradish peroxidase, washed with PBST and visualized with chemilu minescence reagent on an ImageQuant LAS 4000, Antibodies against cyclin dependent kinase inhibitor 2A, estrogen receptor alpha, and proges terone receptor have been obtained from Santa Cruz Biotechnology, Anti secreted frizzled associated protein 1, anti mouse HRP, anti rabbit HRP antibodies had been purchased from Abcam, Anti glutathione S transferase P was purchased from Cell Signaling Technologies, anti actin was bought from Sigma, and anti leptin was bought from R D Systems. In vivo tumorigenicity assay All procedures involving animals had been performed in compliance with State and Federal law, standards on the US Department of Health and Human Services, and suggestions established by Tulane University Institutional Animal Care and Use Committee, All proto cols had been approved by the Tulane IACUC. SCID beige immuno compromised female ovariectomized mice had been obtained from Charles River Laboratories, Mice had been divided into therapy groups of five animals, with or without the need of estro gen.
MCF7 only, MCF7 plus Ob Ab ASCs, MCF7 plus Ob Ab ASCs, MCF7 plus Ob Ab ASCs, and MCF7 plus Ob Ab ASCs, Where indicated, estradiol pellets were implanted subcutaneously inside the lateral region in the neck. MCF7 cells alone or MCF7 cells in combin ation with ASCs suspended discover more here within a total volume of 50 ul of sterile PBS had been mixed with 100 ul of lowered development element Matrigel, Cells had been injected subcutaneously into the fifth mammary fat pad on both sides. All procedures in animals had been carried out under anesthesia utilizing a mixture of isoflurane and oxygen delivered by mask. Tumor size was measured each and every three days employing digital calipers and calculated as previously described, At necropsy, animals were euthanized by cervical dislocation following exposure to CO2. Tumors have been re moved and frozen in liquid nitrogen or fixed in 10% neutral buffered formalin and paraffin embedded for fur ther evaluation.