The cell lines used in this study were obtained from the American Type Culture

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1 Supplementary materials and methods Cell culture and drug treatments The cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and grown as previously described. Lung cancer cell lines A549 and H1355 were cultured on Dulbecco s modified Eagle s medium (DMEM) (GIBCO, Rockville, MD) and RPMI, respectively. CL1-0 and its sub-lines, CL1-5 (human lung carcinoma cell lines with either low or high invasive and metastatic capabilities), were obtained from Dr. Pan-Chyr Yang (1). Cells were either left untreated or were treated with (8-anilino-1-naphthalene sulfonate; ANS) at a final concentration of 200 μm, for 0, 6 and 24 h. Cell lines were maintained at 37 in a 5% CO 2 humidified atmosphere on a medium containing 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD, USA) and 100 ng/ml each of penicillin and streptomycin (Life Technologies, Inc.). Boyden chamber assay, wound healing assay and in vitro cell growth assay Approximately 1.5x10 6 cells/well of complete medium were placed in the upper chamber of Boyden chambers (pore size, 8 μm, GE polycarbonate, PVPF) and a mille-liter of the medium without serum was placed in the lower chamber. After 16 hours of culture, the cells were fixed in methanol for 30 minutes. The filters were washed with PBS and then stained with 20% Giemsas in PBS, for 2 hours. The culture insert model (Ibidi ref: 80209) was used to assess in vitro wound healing (Biovalley, MarnelaValle e, France). Cells were treated with or without 50 and 100 ng of recombinant His-tagged human CCN2(r-hCCN2, BioVendor Laboratory Medicine Inc., Brno, Czech Republic) during culture in DMEM that contained 0.1% horse serum. Cells were seeded on 6 well plates, at a density of 1

2 1x10 5 cells/well, in culture medium and in two separated compartments of the Ibidi insert, according to the manufacturers protocol. Cells on the underside of those analyzed were viewed and counted using a Nikon Ri1 (Digital sight), using a NIS-Elements D 3.0 software (Japan) microscope system. Each experiment was repeated at least three times. For in vitro cell growth assay, the A549 and H1355 transfectants were added to 6-cm dishes that initially contained cells per well. Cells were then trypsinized and resuspended, and cell numbers were counted using a hemocytometer, at varying time points, using a Countess TM Automated Cell Counter (Invitrogen, Carlsbad City, CA). Isolation of RNA, RT-PCR and Real time-pcr Total RNA was isolated using Trizol reagent (Life Technologies, Grand Island, NY), according to the manufacturer s instructions. The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide. Then, cdna was reverse-transcribed from 2 μg total RNA, using random hexamer primers and MMLV-RTase (Promega). Using ABI PRISM 7000 real-time PCR System and TaqMan gene expression probe (Applied Biosystems, Foster City, CA, USA), real-time PCR analysis of GST-M2, CCN2 and GAPDH gene expressions were performed, as have been previously described (Applied Biosystems: assays-on-gene expression products, CCN2 probe: Hs _m1; GST-M2 probe: Hs _g1; GAPDH probe: Hs _m1)(2). The threshold was set above the non-template control background and within the linear phase of target gene amplification, to calculate the cycle number at which the transcript was detected (denoted CT). 2

3 Western blot analysis Cells were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, ) and the concentration was determined using a standard commercial kit (Bio-Rad Lab Ltd., Watford, UK), with bovine serum albumin as a standard. Equal amounts of proteins from each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difuoride membrane (PVDF) (Amersham, RPN303F). The blots were blocked with 5% BSA, for 1h at room temperature, and then probed with rabbit anti-human antibodies against GST-M2 (Santa Cruz, Santa Cruz,CA,USA), GST-M1/M2 (IMGENEX, San Diego, CA) and anti-β-actin (Sigma, AC-40; 1:100000), overnight at 4 C. After three washes, the blots were subsequently incubated with HRP-conjugated secondary antibody, for 1 h, washed again and visualized for chemiluminescence using enhanced luminal reagent (Perkin Elmer, MA, USA). Peptide-affinity purified polyclonal antibody to GST-M1/M2 also recognizes human GST-M2 protein, due to their great similarity. The fold of the images was counted using Image J software (USA, National Institutes of Health (NIH). Construction of GST-M2 plasmid and CCN2 promoter, Transient transfections and luciferase assay The cloning process for GST-M2 expression plasmids was described previously (3). The CCN2 promoter, (-813/+17) luciferase construct (pgl3-ccn2-luc), was as described previously (4). Briefly, 830-basepair (bp) fragments, representing the 5 up stream region of the CCN2 gene (-813 to +17 bp) were generated by PCR. The transcription start site was defined as +1. (GenBank accession number AC099695). The PCR fragment was digested with appropriate enzymes and cloned into pgl3 3

4 basic (Promega). All of the constructs were verified by DNA sequencing and enzyme digestion. For transient transfections and luciferase assay, pcdna/a549 and GST-M2/A549 cells (1 x 10 5 cells per well) were seeded onto 24-well plates and cells were transfected the following day, using JetPei Transfectin reagent (Bio-Rad) with 2 μg of CCN2-Luc, the CCN2 promoter construct. Co-transfection with β-galactosidase control vector was used to normalize, for transfection efficiency. β-galactosidase was measured using Galacto-Light-Plus (Tropix). References 1. Chien W, Yin D, Gui D, Mori A, Frank JM, Said J, et al. Suppression of cell proliferation and signaling transduction by connective tissue growth factor in non-small cell lung cancer cells. Mol Cancer Res. 2006;4: Tang SC, Sheu GT, Wong RH, Huang CY, Weng MW, Lee LW, et al. Expression of glutathione S-transferase M2 in stage I/II non-small cell lung cancer and alleviation of DNA damage exposure to benzo[a]pyrene. Toxicology letters. 2010;192: Weng MW, Hsiao YM, Chiou HL, Yang SF, Hsieh YS, Cheng YW, et al. Alleviation of benzo[a]pyrene-diolepoxide-dna damage in human lung carcinoma by glutathione S-transferase M2. DNA Repair (Amst). 2005;4: Grotendorst GR, Okochi H, Hayashi N. A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor 4

5 gene. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research. 1996;7:

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