Figure 6.

The syndecan-2 cytoplasmic domain induces MMP-7 expression independent of extracellular domain shedding. A, schematic representations of the studied syndecan-2 proteins rat syndecan-2 WT, the L149I non-cleavable mutant (NC), the syndecan-2 mutant lacking the whole cytoplasmic domain (ΔC31), and the L149I non-cleavable mutant lacking the whole cytoplasmic domain (NCΔC31). B, HT-29 cells were transfected with 1 μg of vectors encoding WT syndecan-2 or the ΔC31, NC, or NCΔC31 mutants. Total RNA was extracted, and the expression levels of the target mRNAs were analyzed by RT-PCR. GAPDH was used as a loading control (top left). The expressions of MMP-7 were analyzed by RT-qPCR (bottom left). TCL were subjected to immunoblotting with the indicated antibodies. GAPDH was detected as a control (top right), and quantitative analysis of three independent experiments was performed (bottom right). Data are shown as mean ± S.D. (error bars) (n = 3), normalized to GAPDH expression or total FAK expression. *, p < 0.05 versus SDC2. C, HT-29 cells were transfected with 1 μg of vectors encoding WT syndecan-2 or the NC or NCΔC31 mutants. After the indicated times, the expression levels of the target mRNAs were analyzed by RT-PCR (top), and conditioned media were subjected to slot blotting (SB) with anti-syndecan-2 antibody (bottom). D, HT-29 cells were transfected with 1 μg of vectors encoding WT syndecan-2 or the ΔC31, NC, or NCΔC31 mutants and plated on RTCA CIM-plates. After the indicated times, cell migration was monitored using an xCELLigence system. Representative results from three independent experiments are shown as mean ± S.D. *, p < 0.05 versus SDC2. E, the indicated cells (1 × 10⁵ cells/well) were seeded on soft agar. After 17 days, colonies were stained with 0.005% crystal violet and counted. Shown are representative results from three independent experiments. Data are shown as mean ± S.D. (n = 3). *, p < 0.05 versus SDC2.