Supplementary Table 1. PCR amplification conditions for each primer pair. Primer sequence

Transcription

1 - 1 - Supplementary Tables Supplementary Table 1. PCR amplification conditions for each primer pair Primer sequence FN1 S - CAAAGCAAGCCCGGTTGT AS - CGCTCCCACTGTTGATTTATCTG ITGα2 S - TTAGGTTACTCTGTGGCTGCAATT AS - AGGAGCACCAGCAACAAAGTG TIMP-1 S - GCACAGTGTTTCCCTGTTTATCC AS - GTCCGTCCACAAGCAATGAGT CD44 S - AGCTCCACCTGAAGAAGATTGTACA AS - TTGGTCCATCAAAGGCATTG ICAM1 S - GCCGGCCAGCTTATACACAA AS - TGGCCACGTCCAGTTTCC TGFβ1 S - AAAGATGGAACCCCTCCAATTG AS - CTTTAATTATGTGGTTCCGAAGCA S represents sense. AS represents antisense

2 - 2 - Supplementary Table 2 Relative ECM and adhesion molecule expression in secretory phase primary human endometrial epithelial cells. Symbol UniGene RefSeq Relative Density RPS27A Hs NM_ MMP7 Hs.2256 NM_ FN1 Hs NM_ SPARC Hs NM_ COL11A1 Hs NM_ ADAMTS1 Hs NM_ COL6A3 Hs NM_ COL8A1 Hs NM_ ITGA3 Hs NM_ ITGB1 Hs NM_ CTNND2 Hs NM_ ITGAL Hs NM_ ADAMTS13 Hs NM_ ICAM1 Hs NM_ MMP2 Hs NM_ MMP12 Hs.1695 NM_ MMP1 Hs NM_ TIMP1 Hs NM_ CDH1 Hs NM_ TGFBI Hs NM_ MMP10 Hs.2258 NM_ ADAMTS8 Hs NM_ COL15A1 Hs NM_ COL9A1 Hs NM_ ITGA7 Hs NM_ COL7A1 Hs NM_ COL4A2 Hs NM_ COL6A2 Hs NM_ CD44 Hs NM_ MMP11 Hs NM_ THBS1 Hs NM_ ITGAV Hs NM_ LAMA3 Hs NM_ COL6A1 Hs NM_ COL8A2 Hs NM_ COL18A1 Hs NM_ CSPG2 Hs NM_ ITGAM Hs NM_ ITGA2 Hs NM_ ITGA6 Hs NM_ LAMB4 Hs NM_ CTGF Hs NM_ COL12A1 Hs NM_ LAMA5 Hs NM_ ITGA8 Hs NM_ ITGB5 Hs NM_ ITGB4 Hs NM_

3 - 3 - CTNNA1 Hs NM_ LAMB3 Hs NM_ LAMB1 Hs NM_ Relative expression value represents the mean of two experiments and triplicate hybridizations. Gene expression was normalized to β-actin. Supplementary Table 3. Alterations in extracellular matrix and adhesion molecule expression following IL-11 or LIF treatment of endometrial epithelial cells isolated from mid-secretory phase endometrium. IL-11 LIF (fold change from control) Integrin α TIMP CD ICAM TGFβ Data shows mean of two experiments and triplicate hybridizations and is expressed as fold change from control and represents increase (+) and decrease in IL-11 or LIF treated cells vs. control. Gene expression was normalized to β-actin Optical Density α2 α3 α1 αv α5 α4 β1 β2 β4 β3 β6 αvβ3 αvβ5 α5β1-3 -

4 - 4 - Figure Legend Integrin subunit/dimer profile of primary human endometrial epithelial cells (EEC). Integrins in EEC were determined by Antibody arrays (Chemicon International) as described in the Methods. Equal numbers of viable cells from the treatment groups were assayed by the Antibody arrays. Data represented as mean of triplicate treatment wells ± SEM from three independent experiments. Supplementary Figure 2 A C 0.25 * CONT CONT IL-11 IL-11+ Anti-IL-11 IL-11(ng/ml) 2.0 CONT B LIF (ng/ml) Figure Legend Supplementary Figure 2 Effect of IL-11 and LIF on STAT3 activation in human endometrial epithelial cells. Primary human endometrial epithelial cells (EEC) were treated with (A) IL-11 (1-100ng/ml) (B) LIF (1-100ng/ml) for 15 min. The human endometrial epithelial cell line (HES) was treated with (C) IL-11 (100ng/ml) and neutralizing antibody (anti-il-11, 10μg/ml) or (D) LIF (100ng/ml) and LIF antagonist (LA, 500ng/ml) for 15 min. Cellular protein lysates were subjected to Western blot analysis for phosphorylated (p) STAT3 or STAT3. (A-B) EEC cells (C-D) HES cells. Graphical representation of pstat3/total STAT3 ratios calculated from densitometry on Figure 1A- D respectively. (A-B) Data represents mean of duplicate treatment wells from two independent experiments. (C-D) Data represented as mean of duplicate treatment wells ± SEM from three independent experiments; * p < 5 compared to all other treatment groups. 2.0 * D CONT LIF LIF+ LA - 4 -

5 - 5 - Supplementary methods Extracellular Matrix and Adhesion Molecule Semi-Quantitative Array Method The expression of 113 genes in control and cytokine - treated EEC was profiled using pathway specific Oligo GEArray Human Extracellular Matrix and Adhesion Molecules Microarray (SA Biosciences, MD, USA) according to the manufacturer s instructions. Using the TrueLabeling-AMP 2.0 kit (SA Biosciences), total RNA was used to synthesize cdna. The cdna was further used in the synthesis, labeling and amplification of crna. Purification of crna was performed by a series of washes through spin columns (provided in the True Labeling kit), and quantified by UV spectrophotometry. Five µg of the generated biotin-labeled crna was hybridized to the oligo arrays overnight (18 hrs) at 60 C. The arrays underwent a series of stringent washes, and were subsequently incubated with CDP-Star (SA Biosciences) for the chemiluminescent detection of bound crna. To ensure quantitation within the linear phase of the reaction, membranes were exposed to X-ray film for between 2 sec and 5 min. X-ray films were scanned and analyzed using Quantity One version software (Bio-Rad, Hercules, USA). Relative mrna expression was calculated relative to β-actin. Genes regulated > two fold by either IL-11 or LIF were chosen for verification by quantitative realtime RT-PCR. Antibody Arrays Cell surface integrin protein was assessed by an integrin-mediated cell adhesion array combo kit (Chemicon International) containing 96 wells coated with six mouse anti-human α, five anti-human β integrin subunit monoclonal antibodies and three mouse anti-human α/β integrin dimer monoclonal antibodies. Adhesion assays were performed as per manufacturer s instructions. The cells were harvested using a 1:1 0.12% trypsin-2% EDTA solution (SAFC, St.Louis, USA) and PBS for 5 min and re-suspended in assay buffer at a cell density of 3 x 10 5 EECs/mL medium. Cells were plated in microwells (100 μl/well) and incubated for 90 min at 37 C. The wells were rinsed with assay buffer to remove non-adherent cells. Each well was stained with 100 μl of cell stain solution before rinsing off excess stain solution using deionized water, and extracting the dye. The absorbance reading was taken at 560 nm using a microplate reader (2103 Multilabel Reader; PerkinElmer, Turku, Finland)