Figure 7.
Figure 7. Composition maturation of focal adhesions and matrix remodeling occurs when cellular tension is reduced by 80%. (A) Western blot showing reduced levels of pMLC in U2OS cells in the presence of 1, 5, and 10 µM Y-27632 in comparison with the loading control GAPDH. (B) A bar graph showing the total traction force exerted by U2OS cells (error bars indicate SEM; n = 18, 13, 7, and 9 for WT, 1, 5, and 10 µM of Y-27632, respectively; **, P < 0.01; ***, P < 0.001 with respect to WT). (C) Immunostaining of actin, phosphorylated FAK (FAK-pY397), and Pxn in U2OS cells treated with varying concentrations of Y-27632. The bottom row represents the mean ratio of the background-subtracted FAK-pY397 to Pxn signal at each focal adhesion. Bar, 10 µm. (D) Bar graph of the mean ratio of FAK-pY397:Pxn for WT, 1, 5, and 10 µM of Y-27632 (error bars indicate SEM; n > 500 focal adhesions from at least 10 cells for each condition). (E) The mean intensity (Inten.) of FAK-pY397 or Pxn plotted against the mean intensity of actin at each focal adhesion (FA) for the focal adhesions shown in the bottom row of C. A.U., arbitrary unit. (F) Immunostaining of actin, phosphorylated Pxn (Pxn-pY31), and Pxn in U2OS cells treated with Y-27632. The bottom row represents the mean ratio of the background-subtracted Pxn-pY31 to Pxn signal at each focal adhesion. Bar, 10 µm. (G) Bar graph of the mean ratio of Pxn-pY31:Pxn for WT, 1, 5, and 10 µM of Y-27632 (error bars indicate SEM; n > 500 focal adhesions from at least 10 cells for each condition). (H) The mean intensity of Pxn-pY31 or Pxn plotted against the mean intensity of actin at each focal adhesion for the focal adhesions shown in the bottom row of F. (I) Representative immunostained images of actin, Pxn, and fibronectin in 3T3s treated with varying concentrations of Y-27632. Bar, 30 µm.

Composition maturation of focal adhesions and matrix remodeling occurs when cellular tension is reduced by 80%. (A) Western blot showing reduced levels of pMLC in U2OS cells in the presence of 1, 5, and 10 µM Y-27632 in comparison with the loading control GAPDH. (B) A bar graph showing the total traction force exerted by U2OS cells (error bars indicate SEM; n = 18, 13, 7, and 9 for WT, 1, 5, and 10 µM of Y-27632, respectively; **, P < 0.01; ***, P < 0.001 with respect to WT). (C) Immunostaining of actin, phosphorylated FAK (FAK-pY397), and Pxn in U2OS cells treated with varying concentrations of Y-27632. The bottom row represents the mean ratio of the background-subtracted FAK-pY397 to Pxn signal at each focal adhesion. Bar, 10 µm. (D) Bar graph of the mean ratio of FAK-pY397:Pxn for WT, 1, 5, and 10 µM of Y-27632 (error bars indicate SEM; n > 500 focal adhesions from at least 10 cells for each condition). (E) The mean intensity (Inten.) of FAK-pY397 or Pxn plotted against the mean intensity of actin at each focal adhesion (FA) for the focal adhesions shown in the bottom row of C. A.U., arbitrary unit. (F) Immunostaining of actin, phosphorylated Pxn (Pxn-pY31), and Pxn in U2OS cells treated with Y-27632. The bottom row represents the mean ratio of the background-subtracted Pxn-pY31 to Pxn signal at each focal adhesion. Bar, 10 µm. (G) Bar graph of the mean ratio of Pxn-pY31:Pxn for WT, 1, 5, and 10 µM of Y-27632 (error bars indicate SEM; n > 500 focal adhesions from at least 10 cells for each condition). (H) The mean intensity of Pxn-pY31 or Pxn plotted against the mean intensity of actin at each focal adhesion for the focal adhesions shown in the bottom row of F. (I) Representative immunostained images of actin, Pxn, and fibronectin in 3T3s treated with varying concentrations of Y-27632. Bar, 30 µm.

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