Figure 1.
Detection of FullEGFP and 78Sig constructs and their effects on the expression and shedding of syndecan-1 in transfected HT-1080 cells.
(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.
Table 1.
List of recognition sequences targeted by silencer plasmids used.
Table 2.
List of primers used.
Figure 2.
Effects of FullEGFP and 78Sig on the proliferation and chemotactic migration of HT-1080 cells.
(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).
Figure 3.
Effects of syndecan-1 and -2 gene silencing on the proliferation and migration of HT-1080 cells.
(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.
Figure 4.
Truncated syndecan-1 does not affect the proliferation or the migration of HT-1080 cells if syndecan-2 is silenced.
(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).
Figure 5.
Effects of FullEGFP and 78Sig on the malignancy of HT-1080 cells in vivo.
(A) Size of primary tumours in the footpads of SCID mice on the 24th day after injection of HT-1080 cells expressing EGFP, FullEGFP or 78Sig. Photographs and results of morphometric analysis are shown. *p<0.05 (t-test), #p<0.05 (χ2-test)(n = 5). (B) Histological appearance of lung metastases (arrows) of HT-1080 transfectants. Images do not represent the area percentage of metastases; rather, the only one small EGFP tumour found is shown. HE-stained sections are shown, scale bar: 200 µm. (C) CDK2, phospho-retinoblastoma (at T373 position) and GAPDH immunoblots from HT-1080 primary tumours stably expressing EGFP, FullEGFP or 78Sig. (D) Relative syndecan-2 mRNA levels of the transfectants. Values are expressed as mean±s.d calculated by relative quantification of three independent qRT-PCR results using GAPDH as reference gene and EGFP transfected control as calibrator. *p<0.05 (non-parametric Mann-Whitney test) compared to EGFP control cells. (E) Immunofluorescent staining of methanol-fixed frozen sections by the syndecan-2 specific antibody L-18. Identical exposure times and background corrections were applied Scale bar: 50 µm.
Table 3.
Size of primary tumours on the 24th day after inoculation of stably transfected HT-1080 cells (mm3).
Table 4.
The area percentage of lung metastases.
Figure 6.
Effects of syndecan-1, -2 and -4 overexpression on the proliferation and migration of HT-1080 cells.
(A) Doubling times of stable transfectants were calculated from the log phase of growth curves obtained from SRB colorimetric assays. Values are expressed as mean±s.d. (n = 8). (B) Relative amounts of migrated cells toward ECM proteins in a Boyden chamber. Values are shown as mean±s.d. (n = 5). (A-B) EGFP denotes cells transfected with the empty pEGFP-N1 vector. Sdc-1, Sdc-2 and Sdc-4 refer to syndecan-1, -2 and -4 with no EGFP-tag, respectively, and wt to untransfected wild type (wt) cells. Symbols are *p<0.05 versus EGFP, **p<0.01 versus EGFP, #p<0.05 versus wt, and ##p<0.01 realtive to wt.
Figure 7.
Candidates in the molecular mechanism of the cooperation between syndecan-1 and syndecan-2.
(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 Table S1. (D) Results of densitometry of western blots. Values are expressed as mean±s.d. *p<0.05 by t-test.