Expression levels relative to HAd were determined using the ����Ct method. Histology and IHC Tissue sections (5 ��m) were cut from formalin fixed paraffin embedded (FFPE) blocks. A section for each specimen upon was stained with hematoxylin and eosin (H&E) to confirm the histological features. Unless stated otherwise, all incubations were done at room temperature. Sections were deparaffinized in xylene and hydrated in decreasing concentrations of ethanol. The tissue sections were then subjected to epitope retrieval by steaming in 10 mM sodium citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. Blocking of non-specific sites was achieved by incubation with 5% bovine serum albumin for one hour.

Specimens were then incubated overnight at 4��C with a 1:200 dilution of anti-CDO1 antibody (Sigma-Aldrich). The sections were incubated for one hour with a 1:1,000 dilution of biotinylated anti-rabbit IgG (Jackson ImmunoResearch) followed by a 30-minute incubation with a 1:1,000 dilution of HRP-conjugated streptavidin (Jackson ImmunoResearch). Immunocomplexes were visualized with 3,3-diaminobenzidine tetrahydrochloride (Covance, England). Incubations omitting the primary antibody were used as negative controls for each sample. Quantitation of CDO1 IHC Images from three to five fields of each CDO1 antibody-stained tumor specimen and of its matched control (no CDO1 antibody) were acquired using Nikon Eclipse E400 together with NIS Elements software. To minimize variation, identical exposure conditions were applied to each specimen and its corresponding control.

The CDO1 signal intensity was calculated by NIS Elements for each field of a given tumor sample. The CDO1 expression level was calculated by subtracting the average intensity of all fields of the control slide from the average intensity of all fields of the CDO1-stained slide. DNA extraction and analysis of promoter hypermethylation Genomic DNA was extracted from tumors and from hMSCs before and after each adipogenic induction cycle using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer��s protocol. The optional RNase A treatment was performed. The concentration and purity of DNA was determined by Nanodrop spectrophotometer. A total of 1 ��g of genomic DNA was bisulfite-converted and purified using the EZ Methylation-Direct Kit (Zymo Research).

In all, 9 ng of converted DNA was used as the template in a 25 ��L methylation-specific quantitative PCR reaction.27 Briefly, Cilengitide the reaction contained 1��TaqMan? Universal Master Mix (Life Technologies), 0.2 ��M fluorescent probe, and 1.2 pmol each primer (sequences in Supplementary Table 1 from Ref. 24). The thermal cycling protocol was as follows: 95��C (10 minutes) followed by 50 cycles of 95��C (15 seconds), 60��C (1 minute), and 72��C (15 seconds). Amplification of the unmethylated ��-actin promoter was used as the reference gene (sequences in Supplementary Table 1).