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1 Supporting Information for Microcystin-LR promotes melanoma cell invasion and enhances matrix metalloproteinase-2/-9 expressions mediated by NF-кB activation Xu-Xiang Zhang 1, Ziyi Fu 2, Zongyao Zhang 1, Chen Miao 2, Pengfei Xu 2, Ting Wang 2, Liuyan Yang 1, Shupei Cheng 1 1 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing , China; 2 State Key Laboratory of Reproductive Medicine, Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing , China. Submitted to Environmental Science & Technology Total Pages: 13 Number of Tables: 1 Number of Figures: 8 S1

2 Contents: Table S1 Real-time reverse transcription PCR primers of mmp-2, mmp-9 and β-actin genes. Figure S1 Effects of microcystin-lr exposure on invasion ability of hepatocarcinoma cell HepG2, gastric cancer cell SGC7901, and melanoma cell MDA-MB-435. Figure S2 Effects of microcystin-lr exposure on viability of melanoma cells (MDA-MB-435s). Figure S3 Scatter plot of the 84 metastasis-related genes detected by using the Tumor Metastasis RT² Profiler PCR Array. Figure S4 Fold changes of the 84 metastasis-related genes detected by using the Tumor Metastasis RT² Profiler PCR Array. Figure S5 Activity of MMP-2/-9 in MDA-MB-435 cells treated with Microcystin-LR. Figure S6 Translocation of NF-κB (p65) in melanoma cells (MDA-MB-435) treated with microcystin-lr. Figure S7 Role of NF-κB in the microcystin-induced MMP-2/9 over-expression. Figure S8 OATP1B1 and OATP1B3 (organic anion transporting polypeptides) genes in MDA-MB-435 cells detected by using reverse transcription-pcr (RT-PCR). List of References S2

3 Table S1 Real-time reverse transcription PCR primers of mmp-2, mmp-9 and β-actin genes. Gene name mmp-2 mmp-9 β-actin Primers Forward: 5 - GAG AAC CAA AGT CTG AAG AG-3 Reverse: 5 -GGA GTG AGA ATG CTG ATT AG-3 Forward: 5 - TGC CCG GAC CAA GGA TAC AG-3 Reverse: 5 - TCA GGG CGA GGA CCA TAG AG-3 Forward: 5 -TGA CGT GGA CAT CCG CAA AG-3 Reverse: 5 - CTG GAA GGT GGA CAG CGA GG-3 S3

4 Figure S1 Effects of microcystin-lr exposure on invasion ability of hepatocarcinoma cell HepG2, gastric cancer cell SGC7901, and melanoma cell MDA-MB-435 revealed by transwell chamber assay (A) and statistical analysis (B). (A) The cells were seeded in a 24-well ECM-coated transwell chamber and incubated at 37 C for 72 h with 25 nm microcystin-lr. The cells with invasive capacity were stained with crystal violet and captured of three randomly selected regions under light microscopy ( 100). Representative data from one of three independent experiments are shown. (B) Cells were lysed by extraction buffer before collection and the optical density (OD) were detected at 570 nm for each sample. The value represents the mean ± SD from three different experiments. *P < 0.05 compared with the control cells without microcystin-lr treatment. S4

5 Figure S2 Effects of microcystin-lr exposure on viability of melanoma cells (MDA-MB-435s). The cancer cells were plated in 200 µl L15 media ( cells per well) containing different concentrations of microcystin-lr in a 96-well plate. After the cells were incubated at 37 o C (5% CO 2 ) for different time, the media were discarded and each well was then added with 20 µl fresh MTT S5

6 (3-[4,5-dimethylthiazole]-2,5-diphenyltetrazolium bromide, 5 mg/ml, dissolved in fetal bovine serum) and placed in darkness (37 o C, 5% CO 2 ) for 3-4 h. The liquid was removed to dry the well and formazan (MTT metabolic product) in each well was resuspended in 200 µl DMSO before the plate was placed on shaking table for 10 min to thoroughly mix the formazan into the solvent. Optical density was read at 570 nm. Each treatment group contained five replicate wells and the experiments were repeated three times. * P < 0.05 compared with the control cells without microcystin-lr treatment. S6

7 Figure S3 Scatter plot of the 84 metastasis-related genes detected by using the Tumor Metastasis RT² Profiler PCR Array (Catalog No. PAHS-028A) (SABiosciences, USA). Cells treated with 25 nm microcystin-lr for 24 h were chosen to investigate the relative expressions of the metastasis-related genes against the control (no microcystin-lr treatment). Average values were obtained from two PCR arrays with cdna from each of the treated and control cells. The black line indicates fold changes (2 - Ct ) of 1. The pink lines indicate the desired fold-change in gene expression threshold (± 2.0). S7

8 Figure S4 Fold changes of the 84 metastasis-related genes detected by using the Tumor Metastasis RT² Profiler PCR Array (Catalog No. PAHS-028A) (SABiosciences, USA). Cells treated with 25 nm microcystin-lr for 24 h were chosen to investigate the relative expressions of the metastasis-related genes against the control (no microcystin-lr treatment). The fold changes of gene expression were calculated using the method of Livak and Schmittgen (1). The differentiated expressed genes were considered the ones with the ratio of average expression values between the treated cells and the control greater than 2.0 (2). S8

9 Figure S5 Activities of MMP-2/-9 in MDA-MB-435 cells treated with Microcystin-LR. After 24h treatment with the indicated concentrations of microcystin-lr, MDA-MB-435 cells were removed and the supernatants were used for SDS-PAGE gelatin zymography analysis of MMP-2/-9. The gelatin zymography assay was conducted in triplicate and the visualized bands indicating the presence of a protein with gelatinolytic activity were analyzed by the BioSens Gel Imaging System (BIOTOP, China). *P < 0.05 compared with the control cells without microcystin-lr treatment. S9

10 Figure S6 Translocation of NF-κB (p65) in melanoma cells (MDA-MB-435) treated with microcystin-lr. After the cells were treated with microcystin-lr for 24 h, nuclear and cellular proteins were separately extracted and the protein levels of cytosolic NF-кB and nuclear NF-кB were determined by Western blotting using specific anti-p65 antibody. The nuclear translocation activity of NF-кB was determined by the ratio of p65 (nucleus):p65 (cytosol). The experiments were repeated three times and the blots were visualized by enhanced chemiluminescence (Thermo, USA) and analyzed using a scanning densitometer with the molecular analysis software (BioSens Gel Imaging System, BIOTOP, China). *P < 0.05 compared with the control. S10

11 Figure S7 Role of NF-κB in the microcystin-induced MMP-2/-9 over-expression. MDA-MB-435 cells were separately incubated with JSH-23 (10 µm), microcystin-lr (25 nm) and JSH-23 (10 µm)/microcystin-lr (25 nm). After centrifuging to remove the cells, the supernatants were harvested for SDS-PAGE gelatin zymography of MMP-2/-9. The gelatin zymography assay was conducted in triplicate, and the visualized bands indicating the presence of a protein with gelatinolytic activity were analyzed by the BioSens Gel Imaging System (BIOTOP, China). *P < 0.05 compared with the control cells without microcystin-lr treatment. S11

12 Figure S8 OATP1B1 and OATP1B3 (organic anion transporting polypeptides) genes in MDA-MB-435 cells (435S) detected by using reverse transcription-pcr (RT-PCR). The PCRs were conducted following Janneh et al. (3). The length of target gene was 358 bp for OATP1B1 and 286 bp for OATP1B3. Colonic cancer cells (DLD-1) known to contain OATP1B1 and OATP1B3 genes was used as positive control cells (4,5). GAPDH gene was used as positive control gene and ddh2o was used as the blank control for the RT-PCRs. PCR products were further confirmed by DNA sequencing after purification. S12

13 References (1) Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using realtime quantitative PCR and the 2 C(T) method. Methods 2001, 25(4), (2) Fernandes, B.F.; Di Cesare, S.; Neto Belfort, R.; Maloney, S.; Martins, C.; Castiglione, E.; Isenberg, J.; Abourbih, D.; Antecka, E.; Burnier, M.N.J. Imatinib mesylate alters the expression of genes related to disease progression in an animal model of uveal melanoma. Anal. Cell. Pathol. 2011, 34, (3) Janneh, O.; Hartkoorn, R.C.; Jones, E.; Owen, A., Ward, S.A.; Davey, R.; Back, D.J.; Khoo, S.H. Cultured CD4T cells and primary human lymphocytes express hoatps: intracellular accumulation of saquinavir and lopinavir. Br. J. Pharmacol. 2008, 155, (4) Ichihara, S.; Kikuchi, R.; Kusuhara, H.; Imai, S.; Maeda, K.; Sugiyama, Y. DNA methylation profiles of organic anion transporting polypeptide 1B3 in cancer Cell lines. Pharm. Res. 2010, 27, (5) Obaidat, A.; Roth, M.; Hagenbuch, B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer. Annu. Rev. Pharmacol. Toxicol. 2012, 52, S13