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Figure 2
Cell-Matrix Adhesions Disassemble near the Contact upon Collision
(A) Outline of colliding cells showed in zoom of contact area between two cells. Cells outlined in cyan and magenta. The cell labeled in cyan is expressing membrane RFP (mRFP) (red) and GFP-FAK (green).
(B and C) Total area (B) and average size (C) of CMAs near the contact upon a collision. 0 min = first frame in contact. n = 15 cells. (B insert) Total area of CMAs near the contact as cells separate. 0 min = last frame in contact n = 3 cells.
(D) Disassembly rate of CMAs as indicated. Leading edge, n = 24; contact, n = 27.
(E and K) Immunocytochemistry on cells in contact against p-paxillin and vinculin (E) or cells expressing FAK-GFP (K) as indicated. Bottom: color indicating fluorescence intensity.
(F) Schematic illustrating how regions of interest were defined for analysis. Contact region illustrated in blue and free edge in pink.
(G–J, L, and M) Total area and length of CMAs using the markers indicated. n = 80 cells for (G) and (H); n = 76 cells for (I) and (J); n = 16 cells for (L) and (M).
(N–Q) CMA polarity determined by ratio of total area (or length) of p-paxillin (N and O) or vinculin (P and Q) labeled CMAs in free edge over contact or leading edge over trailing edge.
Scale bars, 20 μm. Line graphs and box graphs show mean, error: ± SEM. ∗∗∗p ≤ 0.001. All Mann-Whitney test. See also Figure S2. |
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Figure 5
Src/FAK Inhibition Increases Cell-Matrix Adhesions near the Contact
(A and I) Immunocytochemistry against vinculin as indicated. Zoom of region of contact and free edge region shown in black boxes. Vinculin shown as green in merged image with Hoescht (blue) and cell outlines. Vinculin alone in zoom colored according to fluorescence intensity.
(B–E) Length and total area of vinculin labeled CMAs. Control, n = 33; PF-573228, n = 24; SU6656, n = 36 cells.
(F–H) Phalloidin staining and quantification as indicated. Arrows indicate phalloidin staining; dotted line indicates contact between adjacent cells.
(I) Immunocytochemistry against p-paxillin as indicated. Zoom of region of contact shown in black box. p-paxillin shown as green in merged image with phalloidin (red), Hoescht (blue), and cell outlines. P-paxillin alone in zoom colored according to fluorescence intensity.
(J) Total CMA area per cell near the contact. Control, n = 52; N-cadh BA, n = 50; N-cadh BA + Src Y527F, n = 49 cells.
Scale bars, 20 μm. Bar graphs show mean, error bars are ±SEM. ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ∗p ≤ 0.05. All Kruskal-Wallis tests. See also Figure S4. |
