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. 2021 Feb 26;296:100481. doi: 10.1016/j.jbc.2021.100481

Figure 4.

Figure 4

Transmembrane protein 2 (TMEM2) is required for the efficient formation of focal adhesions (FAs) in cells migrating on hyaluronan (HA)-containing substrates.A, representative images of in situ HA degradation and FA formation by U2OS cells migrating on Col1/FA-HA mixed substrates (green). U2OS cells were either treated with control siRNA (a, b, and c) or siRNA to TMEM2 (d, e, and f). Cells were double stained for vinculin (red) and F-actin (blue). (a and d) in situ HA degradation patterns; (b and e) distribution of vinculin-immunoreactive FAs in relation to sites of HA degradation; and (c and f) distribution of FAs and F-actin. The scale bar represents 10 μm. B, FA formation in cells at the forefront of migration. U2OS cells migrating on Col1/HA mixed substrates were immunostained for vinculin. Migration assays were performed with parental U2OS cells treated with control siRNA (si-Ctr; left panels), parental U2OS cells treated with siRNA against human TMEM2 (si-hTMEM2; middle panels), and mouse TMEM2-transfected U2OS cells treated with siRNA against human TMEM2 (si-hTMEM2 + mTMEM2; right panels). The scale bar represents 5 μm. C, FA size distribution in cells at the forefront of migration as shown in B. Histogram was derived from the measurements of >300 FAs per condition pooled from triplicate experiments. D, average size of FAs in cells at the forefront of migration. Data represent mean ± SD (number of FAs analyzed: >300 per condition pooled from three independent experiments). ∗∗∗p < 0.001; n.s., not significant by one-way ANOVA with Bonferroni's multiple comparison test.