Supplementary Data

Fold change compared with UCB CD133+ cells 10 15 20 25 30 35 -5 0 5 COL4A1 CD68 PLAUR IL8 CXCR4 PE ADFP 13.4% 14.1% TFGBI PE NUPR - MMP12 > JUNB 7.7% SPP1 HDAC1 Fold change compared with UCB CD133+ cells -400 -300 -200 -100 100 200 [Supplementary Figure1] 0 CRABP AIF Rutella S et al. Supplementary Materials and Methods Real-time PCR Real-time quantitative PCR (qPCR) was performed on mRNA samples isolated from either endometrial cancer-derived CD133+ cells or umbilical cord blood CD133+ cells with the RNeasy plus Mini Kit (QIAGEN, Hilden, Germany), as already detailed. Quantitative PCR for a selected group of genes was performed on the iCycler iQ system (Bio-Rad, Hercules, CA). Primer sets were designed using the Beacon Design Software (Version 3) and the sequences available in the GeneBankTM data base. The specific oligonucleotide primer sequences are detailed in Supplementary Table 8. Complementary DNA (cDNA) was prepared starting from 1μg of total RNA using the iScrypt cDNA Synthesis Kit, which contains RNase H + MMLV reverse transcriptase random primers and 5x reaction mixture (Bio-Rad), according to the manufacturer’s instructions. Reactions were conducted in the PTC-0200 DNA Engine (MJ RESEARCH). Amplification was carried out in a total volume of 25μl containing 0.3μM of each specific primer, 12,5μl 2X SYBR Green Master Mix (100mM KCl, 40mM Tris-HCl, pH 8.4, 0.4mM of each dNTP, iTaq DNA polymerase, 6mM MgCl2, SYBR Green I, 20nM fluorescein, stabilizers; Bio-Rad) and 2μl of diluted cDNA. The PCR reactions were cycled starting with a 3-minute template denaturation step at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Standard curves were generated using a serial dilution of the initial amount of control cDNA, to determine the range of template concentration, which showed a good linearity and efficiency for the different reactions. Melt curves of the reaction products were also generated to assess the specificity of the measured fluorescence. Samples were run in triplicate and the mean of threshold cycles (Ct) for each specimen was used to obtain the fold change of gene expression, following the equation: Fold change = 2-∆(∆Ct) where ∆Ct = Ct of the specific gene - Ct of the housekeeping gene; and ∆(∆Ct)= ∆Ct of the specimen - ∆Ct of the control (i.e., umbilical cord blood CD133+ cells). A sample with a fold change equal to 1 represents a sample that has the same expression level as the reference control for a target gene. Calculations were performed with the Excel spreadsheet RelQuant (Bio-Rad, last updated January 2004). Target gene expression was normalized to the geometric mean of the expression of the 3 most stable housekeeping genes in the tissues analysed (1), namely, GAPDH, transferring receptor (TFRC), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ polypeptide (YWHAZ). Supplementary references 1. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:research0034.1-0034.11.

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