List of recognition sequences targeted by silencer plasmids used.

<p>S1miRNA-a and -b are specific for syndecan-1, S2miRNA-a and -b target syndecan-2.</p

Candidates in the molecular mechanism of the cooperation between syndecan-1 and syndecan-2.

<p>(A) Results of pRTK array. Relative extents of IGF1R and Axl phosphorylation in HT-1080 transfectants. Values are expressed as mean±s.d. *p<0.05 versus control EGFP cells (n = 3). (B and C) Representative immunoblots from HT-1080 cells stably expressing EGFP, FullEGFP, 78Sig or Sdc-2. Antibodies used are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039474#pone.0039474.s002" target="_blank">Table S1</a>. (D) Results of densitometry of western blots. Values are expressed as mean±s.d. *p<0.05 by <i>t</i>-test.</p

The area percentage of lung metastases.

<p>Mice were sacrificed on the 50th day after injection of HT-1080 cells into their foot pads expressing EGFP, FullEGFP or 78Sig. Area percentage referring to the area fractions of lung metastases were calculated by morphometrical analysis of hematoxylin-eosin stained sections of the lungs in 5 random planes.</p

Effects of FullEGFP and 78Sig on the proliferation and chemotactic migration of HT-1080 cells.

<p>(A) Doubling times were calculated from the log phase of growth curves (B) obtained from the SRB colorimetric assay. Values are expressed as mean±s.d. (n = 8). (C) CDK2, phospho-retinoblastoma (at T373 position) and GAPDH immunoblots of HT-1080 cells transfected with EGFP, FullEGFP or 78Sig. (D) Representative fields of cyclin E immunocytochemistry (red) on cultured HT-1080 cells transfected with EGFP, FullEGFP or 78Sig. Scale bar: 20 µm. (E) Relative amounts of migrated cells toward ECM proteins in a Boyden chamber after transfection with EGFP, FullEGFP or 78Sig. Values are shown as mean±s.d. (n = 5), *p<0.05 and **p<0.01. (F) Syndecan-2 and GAPDH immunoblots of cultured HT-1080 transfectants. Sdc-2 stands for syndecan-2 transfection. (G) Results of flow cytometry after immunofluorescent staining of stable transfectants using the ZMD.308 antibody for syndecan-2. (H) Immunofluorescent staining of methanol-fixed cells by the syndecan-2 specific antibody L-18 (red). Identical exposure times and background corrections were applied. Scale bar: 50 µm. For (E and G) nuclei were counterstained with DAPI (blue).</p

Truncated syndecan-1 does not affect the proliferation or the migration of HT-1080 cells if syndecan-2 is silenced.

<p>(A) Detection of transcripts of the 78Sig construct in double transfectants after Geneticin selection by RT-PCR using the EGFP forward and the SDC1-CD reverse primers that produce a 550 bp long amplicon. Agarose electroforetograms the PCR products of these primers. GAPDH was used as positive control. S2miRNA-a + EGFP: the S2miRNA-a was co-transfected with the EGFP control vector. S2miRNA-a +78Sig: S2miRNA-a was co-transfected with the 78Sig vector. (B) Cell sorting results after immunofluorescent staining of double transfectants using the M-140 antibody specific for syndecan-2. (C) Doubling times of double transfectants. Values are expressed as mean±s.d. (n = 8). (D) Relative amounts of migrated HT-1080 stable transfectants in 48-well Boyden chamber. Chemoattractants were ECM proteins. Values are expressed as mean±s.d. *p<0.05 and **p<0.05 versus LacZmiRNA cells (n = 5).</p

Size of primary tumours on the 24th day after inoculation of stably transfected HT-1080 cells (mm³).

<p>HT-1080 cells transfected with the corresponding plasmid construct were injected into the foot pads of 5 mice. The volume of primary tumours was measured for all mice.</p

Detection of FullEGFP and 78Sig constructs and their effects on the expression and shedding of syndecan-1 in transfected HT-1080 cells.

<p>(A) FullEGFP represents the full-length syndecan-1/EGFP coding construct; 78Sig denotes the truncated syndecan-1 variant. ED, ectodomain; TM, transmembrane domain; CD, cytoplasmic domain. (B) Syndecan-1/EGFP fused proteins were detected by fluorescent confocal laser microscope on living HT-1080 cells 24 h after transfection. Scale bar: 10 µm. (C) Immunofluorescent staining of transfected HT-1080 cells by GFP specific monoclonal antibody on paraformaldehide-fixed and Triton X-100 permeabilised cells. Scale bar: 10 µm. (D) The mRNA expression of endogenous and recombinant syndecan-1 was examined by qRT-PCR with primer pairs specific for the cytoplasmic domain (SDC1-CD) and the ectodomain (SDC1-ED) or EGFP cDNA (EGFP). Latter one detects transcrips from all three plasmids thus provides information on transfection efficiency. In the course of relative quantification GAPDH served as reference gene, and the EGFP transfected sample as control. Results are expressed as mean±s.d. (n = 3), *p<0.05 versus control EGFP cells. Note, that the SDC-ED primers do not recognize the 78Sig cDNA, thus they detect only endogenous syndecan-1 in 78Sig transfectants. (E) Relative syndecan-1 protein levels in the lysates and in the media of cell cultures by CD138 ELISA that detects syndecan-1 with an ectodomain specific monoclonal antibody (n = 3), *p<0.05. (F) Immunofluorescent staining of transfected HT-1080 cells by syndecan-1 ectodomain specific monoclonal antibody B-B4 on methanol-fixed cells. Identical exposure times and background corrections were applied. Scale bar: 50 µm.</p

List of primers used.

<p>F: forward primer, R: reverse primer.</p

Effects of syndecan-1 and -2 gene silencing on the proliferation and migration of HT-1080 cells.

<p>(A) Relative mRNA levels of syndecan-1 (SDC1) and syndecan-2 (SDC2) in HT-1080 cells transfected with artificial microRNA coding plasmids, specific for β-D-galactosidase (LacZ), syndecan-1 (S1miRNA-a and -b) or syndecan-2 (S2miRNA-a and -b). Values are expressed as mean±s.d calculated by relative quantification of three independent qRT-PCR results using GAPDH as reference gene and LacZmiRNA control as calibrator. (B, C) Results of flow cytometry after immunofluorescent staining of stable transfectants using the B-B4 antibody for syndecan-1 or the M-140 antibody for syndecan-2. (D) Doubling times of stable transfectants. Values are expressed as mean±s.d. (n = 8). (E) Relative amounts of migrated HT-1080 stable transfectants in a 48-well Boyden chamber. The chemoattractants were ECM proteins. Values are expressed as mean±s.d. (n = 5), *p<0.05 versus control LacZmiRNA cells.</p