SUPPLEMENTARY INFORMATION

Microarray Experiments (technical details)

As a control, a fluor-reverse approach was used, in which each sample was labeled twice by inverting the Cy3 and Cy5 dyes (dye swap) to reduce systematic color bias and to assess gene expression reproducibility. RNAs from untreated and treated cells were hybridized together on 2 different cDNA microarrays spotted with 4318 and 4155 unique sequence-verified human cDNA clones, respectively, from the I.M.A.G.E. Consortium (Research Genetics, Invitrogen, Carlsbad, CA, USA). The detailed production of the cDNA microarray slides has been previously described (De Cecco et al., 2004). Each clone was spotted in triplicate. Hybridizations were carried out in a hybridization station (Genomic Solutions, Ann Arbor, MI, USA), slides were scanned using the GenePix 4000A microarray scanner and the resulting images were analyzed using the GenePix v.3.0 software (Axon Instruments, Union City, CA, USA) to quantify the levels of Cy3 and Cy5 fluorescence. The GenePix Pro raw data files were processed using the GenePix post-processing program GP3 (Fielden et al., 2002) to filter and normalize the data to obtain reliable Cy3 and Cy5 ratios of each cDNA target. Data analysis was performed using BRB-ArrayTools v3.1 developed by Dr. Simon and Lam.

Analysis of microarray results

The microarray results were analyzed using Expression Analysis Systematic Explorer (EASE) (Hosack et al., 2003), a software application that provides statistical methods for discovering biological themes within gene lists. The statistical significance of over-represented category is provided by an EASE score generated by a jackknife iterative resampling of Fisher exact probabilities, with Bonferroni multiple testing correction. The EASE score indicates increasing confidence in overrepresentation as the score decreases toward zero. Gene categories with an EASE score of less than 0.05 were determined to be significantly over-represented.

Real time–quantitativePCR

Total RNA was reverse-transcribed using the High-Capacity cDNA Archive Kit and following manufacturer’s instructions. TaqMan TM reactions were carried out in triplicate on an ABI PRISM 7700 machine, using the Assays-on-Demand Gene Expression Product available for PLAB (Hs00171132_m1) and GAPDH as housekeeping gene (Applied Biosystems, Foster City, CA, USA). Data analysis was performed using the Sequence Detector v1.9 software.

RT-PCR oligonucleotide primer sequences

The amplification reaction was initiated by incubation of PCR samples at 94°C for 2 min, followed by cycles of 94°C for 30 s, 54°C for 45 s and 72°C for 45 s, and a final extension for 10 min at 72°C. For each primer set, an increasing number of PCR cycles were performed with otherwise fixed conditions to determine the optimal number of cycles to be used. The number of cycles was 30 for PLAB amplification, 35 for COL3A1, 40 for CYR61, IFTM2, IFTM3, and DHFR, and 20 for actin. The PCR products were subjected to electrophoresis in agarose gel and visualized by UV after ethidium bromide staining.

PLAB forward primer, 5’ACGCTGAATGGCTCTCAGAT-3’; PLAB reverse primer, 5’TGTTCGAATCTTCCCAGCTG-3’; Cyr61 forward primer, 5’CGAGGTGGAGTTGACGAGAAAC-3’; Cyr61 reverse primer, 5’AGGACTGGATCATCATGACGTTCT-3’; IFTM2 forward primer, 5’AATGCCAGGAAGAGGAAACT-3’; IFTM2 reverse primer, 5’GCCATTGTAGAAAAGCGTGT-3’; IFTM3 forward primer, 5’GTGCACTTTATTGAATGCC-3’; IFTM3 reverse primer, 5’CTCCAACTTCCATTCCTCG-3’; COL3A1 forward primer, 5’GTCTGGACCAAAAGGTGATG-3’; COL3A1 reverse primer, 5’GTCCACTGGTTCCATCTTTG-3’; DHFR forward primer, 5’GTAGAAGGTAAACAGAATCTG-3’; DHFR reverse primer, 5’AGAACACCTGGGTATTCTGG-3’; -actin forward primer, 5’GAAATCGTGCGTGACATTAAG-3’; -actin reverse primer 5’CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC-3’.

Supplementary references

De Cecco L, Marchionni L, Gariboldi M, Reid JF, Lagonigro MS, Caramuta S et al. (2004). Gene expression profiling of advanced ovarian cancer: characterization of a molecular signature involving fibroblast growth factor 2.Oncogene23: 8171-8183.

Fielden MR, Halgren RG, Dere E, Zacharewski TR. (2002). GP3:GenePix post-processing program for automated analysis of raw microarray data. Bioinformatics18: 771-773.

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