Figure 3.

A serine residue in the variable region of the syndecan-2 cytoplasmic domain is important for its ability to upregulate MMP-7. A, schematic representations of the studied versions of syndecan-2. The full-length syndecan-2 protein (WT), a mutant lacking the C-terminal EFYA sequence (ΔC4), a mutant lacking half of the variable cytoplasmic domain and EFYA (ΔC14), and a mutant lacking the entire cytoplasmic domain (ΔC31) are shown (top). Cells were stably transfected with the indicated mutant constructs, as described under “Experimental procedures.” Total RNA was extracted, and the expression levels of syndecan-2 and MMP-7 were analyzed by RT-PCR (middle) and RT-qPCR (bottom). Data are shown as mean ± S.D. (error bars) (n = 3), normalized to GAPDH expression. *, p < 0.05. B, the indicated cells were lysed with luciferase lysis buffer, and luciferase activity and β-galactosidase activity were measured. Data are shown as mean ± S.D. (n = 3). *, p < 0.05. C, the indicated cells were seeded on soft agar. After 17 days, colonies were stained with 0.005% crystal violet and counted. Data are shown as mean ± S.D. (n = 3). *, p < 0.05. D, schematic representation of the studied versions of the syndecan-2 cytoplasmic domain. Shown are the WT syndecan-2 cytoplasmic domain and mutants in which serines 197 and 198 were changed to alanine and glutamic acid, respectively (AE); glutamic acid and alanine, respectively (EA); alanine and alanine (AA); and glutamic acid and glutamic acid (EE) (top). Cells were stably transfected with the indicated mutant constructs. After 24 h, the expression levels of the target mRNAs were analyzed by RT-PCR and RT-qPCR (bottom left). TCL were subjected to immunoblotting with the indicated antibodies, and quantitative analysis of three independent experiments was performed (bottom right). Quantitative analysis of three independent experiments was performed and normalized to GAPDH or total FAK expression. Data are shown as mean ± S.D. (n = 3). *, p < 0.05. 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. Data are shown as mean ± S.D. (n = 3); *, p < 0.05.