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First published online 30 September 2008
doi: 10.1242/jcs.027201

Journal of Cell Science 121, 3393-3402 (2008)
Published by The Company of Biologists 2008
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Reduced tumorigenesis in mouse mammary cancer cells following inhibition of Pea3- or Erm-dependent transcription

Virginie Firlej1,*, Franck Ladam1,*, Guillaume Brysbaert2, Patrick Dumont1, François Fuks3, Yvan de Launoit1, Arndt Benecke2 and Anne Chotteau-Lelievre1,{ddagger}

1 UMR 8161, Institut de Biologie de Lille, CNRS Universités de Lille 1 and 2, Institut Pasteur de Lille, IFR 142, BP 447, 1 rue Calmette, 59021 Lille Cedex, France
2 Institut des Hautes Études Scientifiques and Institut de Recherche Interdisciplinaire, CNRS USR3078, Université de Lille 1, 35 route de Chartres, 91440 Bures sur Yvette, France
3 Laboratoire d'Epigénétique du Cancer, Faculté de Médecine, Université Libre de Bruxelles, CP 614, 808 Route de Lennik, 1070 Brussels, Belgium


Figure 1
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  		Fig. 1. Quantification of siRNA-mediated pea3 and erm knockdown. (A,B) Relative mRNA expression levels of pea3 in si pea3 1-2 (A) and erm in si erm 1-2 (B) versus si ctrl transfected MMT cells, assessed by quantitative RT-PCR, 48 hours post transfection. Results are expressed as ratios of mRNA levels of pea3 or erm to cyclophilin (endogenous control standard) (1=si ctrl) and are the mean ± s.d. of three experiments in duplicate. (C) Western blot analysis of Pea3. Extracts of total protein of transfected MMT cells were analyzed using anti-Pea3 and anti-actin (control) antibodies 48 hours post transfection.

 

Figure 2
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  		Fig. 2. pea3 and erm knockdown impairs proliferation, migration and anchorage-independent growth ability of MMT cells. (A,B) Proliferation assays. si pea3 1-2 and si ctrl (A) or si erm 1-2 and si ctrl (B) transfected MMT cells were trypsinized and counted with a cell counter every day. Data are the mean ± s.d. of a representative experiment performed in triplicate. Experiments were reproduced four times in triplicate and gave the same results for each assay. (C) Migration assay. 24 hours post transfection, si pea3 1-2, si erm 1-2 or si ctrl transfected cells were seeded into the upper part of a Boyden chamber. After 18 hours, cells which have migrated through the membrane are counted. Experiments were done at least three times. Data are the mean ± s.d. of a representative experiment performed in triplicate. (D) Anchorage-independent growth assay. 24 hours post-transfection, si pea3 1-2, si erm 1-2 or si ctrl transfected cells were mixed with agar 0.65% and cultivated during 15 days. Experiments were done three times. Data are the mean ± s.d. of a representative experiment performed in triplicate.

 

Figure 3
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  		Fig. 3. pea3 and erm knockdown affects morphogenetic capacity of MMT cells. si pea3 1-2, si erm 1-2 and si ctrl transfected MMT cells were seeded at low density on a Matrigel. 3D cultures were treated with HGF/SF (20 ng/ml). After 7 days, cells were stained with Neutral Red and fixed. Whole-mount pictures were taken at 50x (A) or 100x (B) using a microscope. Scale bars, 100 µm (A) or 100X (B). Images are representative of three experiments performed in duplicate.

 

Figure 4
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  		Fig. 4. In vivo tumor growth of MMT cells stably expressing shRNA, MMT Rs, MMT Ri erm, MMT Ri pea3 and MMT Ri pea3/erm. (A) Validation of shRNA-mediated pea3 and erm knockdown. Relative mRNA expression levels of pea3 in MMT cells infected with pRS-pea3 A (dark gray bars) or pRS-pea3/erm (dark striped bars) and erm in MMT cells infected with pRS-erm (light gray bars) or pRS-pea3/erm (light striped bars) versus control pRS retroviruses, as assessed by quantitative RT-PCR. Results are expressed as ratios of mRNA levels of pea3 or erm to cyclophilin (endogenous control standard) (1=Rs) and are the mean ± s.d. of 3 experiments in duplicate. (B) Tumor growth of the MMT cells stably expressing shRNA. MMT cells infected with pRS-pea3 A, pRS-erm, pRS-pea3/erm or pRS retroviruses were subcutaneously injected into SCID mice. Three days after injection, tumors were measured every 3 days for 15 days. Values represent the mean ± s.d. of one representative experiment (five mice per group) from the three experiments under identical conditions. Error bars indicate + s.d. * P<0.05, ** P<0.01.

 

Figure 5
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  		Fig. 5. Summary of transcriptome analysis and results. Inhibition of either pea3 or erm induces significant changes in the cellular transcriptome of MMT cells. Expression levels of mRNAs from pea3 or erm siRNA-treated cells were individually compared with control oligonucleotide-treated MMT samples by genome-wide microarray analysis. The absolute number of probes detecting statistically significant up- or downregulation following inhibition of either of the two Ets factors is shown to the left of each bar; the maximum positive or negative logarithmic (base two) fold-change is shown to the right. The black to gray gradient indicates positive, and the white to gray gradient negative fold changes in expression. The subset of genes regulated in both cases is also shown ({cap}).

 

Figure 6
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  		Fig. 6. Validation of the microarray experiments by semi- and quantitative RT-PCR. (A) mRNA expression levels of Pea3, Erm, Has2, Hgf, Ascl4, Fgfr1 and cyclophilin (endogenous control standard), in si ctrl (lane 1) versus si pea3 1 (lane 2) and si erm 2 (lane 3) transfected MMT cells, assessed by semi-quantitative RT-PCR. (B) Logarithmic fold-changes for pea3, erm, Fst, Suz12, Has2 and Stip1 genes as determined by quantitative RT-PCR for pea3 (black bars) and erm (gray bars) siRNA-mediated inhibition are shown. Results are expressed as ratios to cyclophilin mRNA levels. Error bars indicate ±s.e.m. of at least three independent biological replicates.

 

Figure 7
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  		Fig. 7. Effects of pea3 or erm siRNA-mediated inhibition on known target genes as determined by microarray analysis. For selected, known pea3- or erm-target genes, the average logarithmic fold-change and standard deviation recovered from the microarray experiments are shown. Black bars indicate the fold-change between pea3 siRNA treated and control siRNA treated MMT cells, gray bars correspond to the erm siRNA-treated versus control-treated cells.

 

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© The Company of Biologists Ltd 2008