Figure 4

Syndecan-2 is a regulator of MDA-MB231 cytoskeleton and behaviour. (A) Specific knockdown of mRNA levels after syndecan-1, −2 or −4 siRNA treatments. mRNA was extracted from cells 48 h after transfection with syndecan-1, syndecan-2, syndecan-4 or control siRNAs. Levels of other syndecan mRNAs were not affected by syndecan-2 knockdown (left). (B) Flow cytometry analysis confirmed loss of cell surface syndecan-2 after silencing syndecan-2 with siRNA. (C) Syndecan-2 depleted cells were stained for F-actin, which showed extensive microfilament bundle formation. Bar = 25 μm. (D) Spread cell areas were increased by syndecan-2 depletion, n ≥ 50 cells per condition. (E) Cadherin-11 containing adherens junctions were visible after syndecan-2 depletion. Bar = 25 μm. (F, G) Type I collagen invasion (F) and degradation (G) were reduced in syndecan-2 depleted cells. Higher magnification images are shown on the lower panels of G. Bar = 100 μm. (H) Cells depleted of each syndecan individually or in combination, by siRNA treatment were stained for focal adhesions (arrows) with a paxillin antibody. Quantification of focal adhesion (FA) size and number was done by one-way ANOVA with Tukey’s post-hoc test. Bar = 25 μm. Error bars = s.e.m. from three independent experiments. ***p < 0.001, n.s.; not significant by two-tailed paired t-test.