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. 2017 Aug 24;113(7):1585–1598. doi: 10.1016/j.bpj.2017.07.027

Figure 6.

Figure 6

N-cadherin and α-catenin recruit vinculin to intercellular junctions. (A) Shown here is Western blot of whole cell lysates of CL-S1 cells transfected with control, N-cadherin (N-cad KD), or α-catenin (α-cat KD) siRNA. (B) Control, N-cadherin, and α-catenin KD cells were plated on VE PDMS and the cell-free area was measured 6 h postsettling (n = 4–7). Cell-free area of N-cad KD cells and α-cat KD were normalized against that of control cells due to variable initial cell density after electroporation between experiments. (C) Shown here are confocal projections of control or α-catenin KD CL-S1 monolayers on VE PDMS immunostained for N-cadherin, vinculin, and nucleus, 4 h postsettling. Quantitative image analysis is given of the proportion of vinculin colocalized with N-cadherin (C′, n = 5–6), and the proportion of vinculin colocalized with paxillin (C″, n = 6–8). (D) Shown here is FRAP of GFP-vinculin-expressing cells at 4–7 h postsettling on different PDMS substrata. The t1/2 of recovery was significantly lower on VE (n = 7) than SE (n = 6) or E (n = 11) substrata. (D′) Given here are immobile fractions on different PDMS substrata. (E) MDCK cells overexpressing GFP, or GFP-tagged full-length vinculin (GFP-vinculin) or vinculin tail (GFP-VT) were plated on VE PDMS and the cell-free area was measured 5 h postsettling (n = 4). MDCK cells overexpressing GFP-tagged to amino acids 1–325 of α-Ecatenin (GFP-αEcat), or GFP-αEcat-tagged to the vinculin head domain (GFP-αEcat-VH) or tail domain (GFP-αEcat-VT), and untransfected cells (n = 5–8) were treated similarly. CL-S1 cells expressing GFP or GFP-vinculin, and untransfected cells, were plated on SE PDMS (n = 3). Overexpression of GFP-vinculin or GFP-αEcat-VT significantly increased cell coalescence in MDCK cells on VE PDMS, but not CL-S1 cells on SE PDMS. (F) Shown here is Western blot of whole cell lysates of different cell lines, immunoblotted for vinculin (vinc) and α-catenin (α-cat). Each well was loaded with the equivalent of 10% of a confluent 3.5 cm dish of cells. ∗p < 0.05, ∗∗∗p < 0.001; n.s., not significant; bars represent SE. To see this figure in color, go online.