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Supplemental Materials SUPPLEMENTARY DATA shRNA-containing lentiviral vectors. The shRNAs for each target genes were designed with the aid of web-based Invitrogen Block-It program. The oligonucleotides containing Fra-1, c-Jun or slug shRNA sequences were inserted into pLV-shRNA vector (Biosettia, San Diego, CA) according to manufacturer’s protocol. The Fra-1 mRNA target sequences are 5’- GGAGACTGACAAACTGGAAGA-3’ and 5’-GGATGGTACAGCCTCATTTCC-3’. The c-Jun mRNA target sequences are 5’-GGAACAGGTGGCACAGCTTAA-3’ and 5’- GGCACAGCTTAAACAGAAAGT-3’. The slug mRNA target sequences are 5’- GCATTTGCAGACAGGTCAAAT-3’ and 5’-GCTCATCTGCAGACCCATTCT-3’. Slug promoter construction. The slug promoter (GeneBank Accession number AB300659) was synthesized by PCR using genomic DNA isolated from MDA-MB-231 cells. The synthesized fragment was subcloned into pGL4-luciferase reporter gene plasmid (Promega, Madison, WI) and sequence confirmed by automated DNA sequencing. To generate AP1 mutant, the AP1 consensus sequence in the slug promoter was destroyed by introducing mutation at nucleotide 1009-1011 (GAC AGA). To determine the promoter activity, wild-type or mutant slug promoter reporter gene construct was transfected into MCF-7, MDA-MB-231, MDA-MB-436 or ZR-75-1 cells for 36 hrs and cell lysates collected to measure luciferase activity using Lipofectamine (Invitrogen, Carlsbad, CA). To determine the effect of Fra-1/c-Jun expression on slug promoter activity, the promoter reporter gene construct was co-transfected with expression vectors encoding Fra-1 and c-Jun into MCF-7 and ZR-75-1 cells for 48 hrs followed by the analysis of luciferase activity. Plasmid containing Renilla Luciferase gene was included during transfection to serve as an internal control and Dual Luciferase Assay System (Promega, Madison, WI) was used for measuring luciferase activity. MDA-MB-231 cells Fra-1 1 0.05 0.15 1 0.07 0.07 relative intensity c-Jun 1 0.9 1.1 1 0.05 0.06 relative intensity pERK ERK EActin control ______1 2 control ______1 2 Fra-1 shRNAs c-Jun shRNAs Fig.S1. The effect of Fra-1 and c-Jun shRNAs on their target expression. MDA-MB-231 cells were infected with lentiviral vector containing luciferase shRNA (control) or Fra-1 or c-Jun shRNA for 4 days. Cells were lysed and cell lysates subjected to immunoblotting to detect Fra-1, c-Jun, phosphor-ERK, ERK2 and Eactin with the respective antibodies. Conclusion: 1) Fra-1 shRNAs can effective downregulate Fra-1 gene expression. 2) c-Jun shRNAs can down regulate both c-Jun and Fra-1 expression. These results suggest that Fra-1 expression requires the presence of c-Jun but not vise versa. MDA-MB-231 0 hr 6 hr 16 hr control Fra-1 shRNA1 c-Jun shRNA2 Fra-1 shRNA1 + c-Jun shRNA2 Fra-1 shRNA1 + c-Jun shRNA2+ U0126 Fig.S2. The effect of Fra-1 and c-Jun shRNAs on cell migration (wound healing). Control MDA-MB-231 cells and MDA-MB-231 cells with Fra-1 or c-Jun knockdown were grown to confluent monolayer in the presence or absence of 5PM U0126 for 2 days. A scratch was made with a fine pipette tip and detached cells were washed away. The cells were fed with medium containing 1% FCS with or without U0126 and images were taken at various times under a microscope. A ) 0.9 0.8 600nm 0.7 0.6 0.5 0.4 * * 0.3 * 0.2 0.1 Cell Migration (OD Cell Migration 0 Fra-1 shRNA1 - + + - - + + murine Fra-1 - - + - - - + c-Jun shRNA2 - - - + + + + murine c-Jun - -- -+ -+ B 0 hr 6 hr 16 hr control Fra-1/c-Jun KD Fra-1/c-Jun KD + Murine Fra-1/c-Jun Fig.S3. Forced murine Fra-1/c-Jun expression rescues cell migration of Fra-1/c-Jun knockdown cells. A. Control MDA-MB-231 and MDA-MB-231 with Fra-1 or c-Jun knockdown were infected with lentiviral vectors encoding murine Fra-1 or c-Jun for 3 days. Cells were detached with 10mM EDTA- containing PBS, then resuspended in serum-free medium and analyzed for cell migration using Transwells. Data are means ± S.E. (*, p<0.01 vs control) B. Fra-1/c-Jun-knockdown MDA-MB-231 cells with forced murine Fra-1/c-Jun expression were grown to confluent monolayer. After a scratch was made, cells were fed with medium containing 1% FCS and images were taken at various times under a microscope. A B 1.2 MDA-MB-231 MDA-MB-436 MDA-MB-231 MDA-MB-436 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 Relative RSV 0.2 Relative RSV Promoter Activity 0 Promoter Activity 0 U0126 - + - - + - Fra-1 shRNA1 - + - + - + - + MEK1(-) - - + - - + c-Jun shRNA2 - - + + - - + + 2 MCF-7 ZR-75-1 C * 1.6 * 1.2 0.8 (F-luc/R-luc) Promoter Activity 0.4 Slug 0 Fra-1 - + - + + - + - + + c-Jun - - + + + - - + + + MEK1(+) -- --+ ----+ Fig.S4. The ERK-Fra-1/c-Jun axis is specifically involved in slug promoter activation. A. MDA-MB-231 and MDA-MB-436 cells were transfected with the RSV promoter luciferase reporter plasmid for 1 day and 5PM U0126 then added to cells for another 2 days. Cells were lysed and cell lysates analyzed for luciferase activity. In a parallel expreriment, the RSV promoter luciferase reporter plasmid was co-transfected into cells with the expression vector containing dominant negative MEK1 [MEK1(-)] for 3 days. Cells were lysed and cell lysates analyzed for luciferase activity. Data are means ± S.E. B. The RSV promoter luciferase reporter plasmid was transfected into control or Fra-1/c-Jun knockdown MDA-MB-231 and MDA-MB-436 cells for 2 days, then lysed and cell lysates analyzed for luciferase activity. Data are means ± S.E. C. The slug promoter luciferase reporter plasmid was co-transfected into MCF-7 and ZR-75- 1 cells with Fra-1/c-Jun plasmid or Fra-1/c-Jun+MEK1(+) for 2 days. Cells were lysed and cell lysates analyzed for luciferase activity. Data are means r SE. (*, p < 0.005 vs control) MDA-MB-231 0 hr 6 hr 16 hr control slug shRNA1 slug shRNA2 slug shRNA2 + murine slug transgene Fig.S5. The effect of slug shRNA on cell migration (wound healing). Control MDA-MB-231 cells and MDA-MB-231 cells with slug knockdown were grown to confluent monolayer. A scratch was made with a fine pipette tip and detached cells were washed away. The cells were fed with medium containing 1% FCS and images were taken at various times under a microscope. In a parallel experiment, murine slug cDNA was forced to be expressed in MDA-MB-231 cells with slug shRNA2 expression and these cells were then analyzed for cell migration by wound healing assay. ) 6000 3 5000 4000 slug knockdown 3000 2000 control Tumor Volume (mm 1000 0 12345678 times after inoculation (week) Fig.S6. In vivo tumor outgrowth of control MDA-MB-231 and MDA- MB-231 cells with slug knockdown. Cells (3 x 106 cells/mouse) were subcutaneously injected at 4th mammary fat pad area of female athymic nude mice (6-7 weeks of age). Xenografts were externally measured in two dimensions using a caliper and tumor volume (V) was calculated by equation: V = (L x W2) x 0.5, where L is the length and W is the width of a xenograft. Student-Newman-Keuls test were used to compare control and slug knockdown groups. Conclusion: there was no statistically significant difference between control and slug-knockdown groups. 7$%/(6*HQHV$OWHUDWHGE\)UDF-XQ.QRFNGRZQ .'FRQWURO .'FRQWURO GENE )ROGFKDQJH *(1( )ROGFKDQJH HAS2 0.29 EFHD1 2.28 ID4 0.31 CDKN1A 2.29 RGMB 0.31 SPANXE 2.31 SNAI2 0.32 LAMB1 2.32 CCNE2 0.37 NDRG4 2.35 OSR1 0.38 NFKBIE 2.36 EVI2A 0.42 LAMB1 2.37 FGB 0.43 TMEM154 2.38 DMBT1 0.43 PRIC285 2.46 CTSC 0.44 FLJ25801 2.49 WNT7B 0.47 TSPYL5 2.51 ID2 0.47 LRP3 2.54 SLC30A1 0.49 TMEM154 2.61 HS.444290 0.5 SPANXD 2.8 PPAP2B 0.5 PSMB8 2.81 LXN 2 CD82 3.07 TNFAIP3 2.03 SPANXA1 3.1 AADAC 2.04 SPANXA1 3.14 NFKB2 2.04 IL32 3.14 SPANXC 2.08 IL1RAPL1 3.16 NFKB2 2.08 CXCL1 3.3 ASB9 2.18 MLLT11 3.5 COCH 2.19 BIRC3 3.68 SPANXB1 2.2 ZC3H12A 3.96 RELB 2.21 CXCL2 4.33 ACTA2 2.21 GPR92 4.41 SPANXA1 2.24 IL8 6.01 CLIC6 2.26 IL8 7.24 TAP1 2.27 CSF2 7.6 KD: knockdown.
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