The KSFrt Apcsi and KSFrt mtApcsi steady cells were seeded a

The KSFrt Apcsi and KSFrt mtApcsi steady cells were seeded at a of 19,000 cells/cm2 and 9500 cells/cm2, respectively, in 24well dishes, and transiently transfected with 2 ug of the reporter construct 2 Luc, BAT Luc or pSAR MT APC using Fugene HD transfection reagent, based on the manufacturers protocol. 2-5 ng of Renilla luciferase was cotransfected, to fix for transfection efficiency. A day after transfection, transfected cells were both left low stimulated o-r stimulated for an additional 24 h. Luciferase assays were done as described previously. TheKSFrt Apcsi andKSFrt mtApcsi stable cells were seeded at a of 24,000 cells/cm2 and 12,000 cells/cm2, respectively, and classy inthe presence or absence of BMP 7 at the levels indicated, to stimulate MAPK phosphorylation osteogenic difference. The medium was changed every 3?4 days. At confluence, when nodules appeared and, ascorbic acid, T glycerol phosphate were included with the culture medium. The amount of mineralization and Investigation of the Alkaline Phosphatase activity was done as previously described. To stimulate chondrogenic differentiation, 300,000 cells were pelleted by centrifugation in a round bottom well of a 96 wellplate and cultured in 250 ul large sugar DMEM, supplemented with 100 U/ml Pen/Strep, 50 ug/ml ascorbic acid, 40 ug/ml proline, 1 mM Pyruvate, 1:100 ITS Premix. During the first two weeks of culture, medium was further enriched with 10 ng/ml TGFB3 and 10?7 Mdexamethasone, Infectious causes of cancer while starting with week 3, 5 mM B and 500 ng/ml BMP 6 glycerol phosphate was put into the medium. The method was replaced every 3?4 times. After 6 months of culture, pellets were set, embedded in paraffin and sectioned. Sections were stained with Toluidine Blue o-r immunostained for collagen II as previously described. Glycosaminoglycan quantification corrected for DNA after 2, 4 and 6 weeks of culture was done as previously described. To stimulate adipogenic differentiation, the KSFrt Apcsi and KSFrtmtApcsi firm cells were seeded at a of 12,000 cells/cm2 and 24,000 cells/cm2, respectively, and cultured in the presence of 25 uM indomethacin after confluence. After 3 months of culture, cells were stained with Oil Red O as described previously. Quantification of adipocytes natural product library was done by counting adipocytes, defined by the presence of at least three lipid drops per cell from nine randomly selected fields for each group. All values represent mean_SEM of several separate triplicate experiments. Differences were examined by one-way analysis of variance. Results were considered significant at p 0. 05. The KSFrt Apcsi cell line is just a good model for studying the role To study the role of the Apc gene in regulating lineage commitment and differentiation of SPC, we generated a cell line with decreased Apc expression by RNA interference applying the 4C3 Frt clone of the KS483 murine host cell line.

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