Skip to main content
. 2017 May 15;8:15321. doi: 10.1038/ncomms15321

Figure 4. YAP regulates focal adhesion/cytoskeleton integrity downstream of RhoA.

Figure 4

(a) Heatmap representing the RNA expression levels by normalized ΔCt values obtained in two qRT–PCR independent replicates of YAP mutant clone C3 as compared with CAL51 WT. (b) Graph: barplot representing mean value±s.d. of up- and downregulated genes with a fold change higher than 2.0 in YAP mutant clone C3 as compared to CAL51 WT. (c) Western blot analysis of the indicated focal adhesion (FA) proteins; n=3. (d) Representative confocal images of WT CAL51 and YAP mutant clones C2a and C3, showing vinculin staining (green). (e) Protein analysis of the indicated cytoskeleton- and FA-associated proteins in WT CAL51 and YAP mutant clones; n=3. (f) Graphs: quantification of cell contact area and Young's Modulus in WT CAL51 and YAP-defective clone C3. Values are shown as median±min/max (n=24, 3 technical replicates, Welch's t-test, ***P<0.001 and **** P<0.0001,). (g) Box: Representative brightfield image of WT CAL51 and C3 clone migrated through ECM-coated transwell membrane (8 μm) and stained with crystal violet after 24 h, quantification is shown in the upper graph. The data represent the mean value±s.d. (12 random fields, 2 technical replicates, **P<0.01 and ***P<0.001, Kruskal–Wallis test followed by post hoc Dunn‘s test). Bottom: qRT–PCR array analysis of genes involved in ECM remodelling in YAP mutant cells (C3) as compared with the control. The threshold was set at 2.0. Right: Representative brightfield image of CAL51 WT and YAP mutant cells C3 grown in three-dimensional (3D) Matrigel for 120 h. (h) Representative confocal images showing the arrangement of F-actin in WT Cal51 and mutant clones C2a and C3 stained with Alexa Fluor 546 Phalloidin. (i) Representative western blot quantification of G-actin/F-actin ratio in WT CAL51 and YAP mutant clones. (j) Representative western blot analysis of the indicated proteins; n=3. (k) Representative confocal image of WT CAL51 cell transfected with EGFP-RhoA-Q63L and stained with anti-YAP antibody (red), Alexa Fluor 647 Phalloidin (white). Image analysis shows the nuclear/cytoplasmic distribution of YAP protein in non-transfected and transfected cells. (l) Confocal images of WT CAL51 and mutant clone C3 transfected with EGFP-RhoA-Q63L and stained with anti-vinculin antibody (red), Alexa Fluor 647 Phalloidin (white).