Figure 3.

ADF/CFL1 Depletion Increases F-Actin Bundles and Focal Adhesions

(A) ADF-null SCCs were assessed by immunoblotting for globular (G) and filamentous (F) β-actin levels following treatment with siNT or siCFL1.

(B) Free barbed-end and F-actin fluorescent visualization of ADF-null SCCs treated as in (A).

(C) Quantification of barbed-end formation of ≥40 cells using ImageJ. Result presented as mean ± SEM (n = 2). ^∗∗∗unpaired t test p value < 0.0001.

(D and E) Focal adhesions were visualized with phospho(p)-paxillin (Tyr118) and vinculin after 48 hr of siRNA treatment. F-actin staining is also shown.

(F–H) Quantification of p-paxillin and vinculin staining, as well as the number of focal adhesions (FAs) per cell based on p-paxillin staining was performed using ImageJ. Box plots display data from min to max. Forty cells quantified for p-paxillin and 63 for vinculin staining in greater than or equal to three fields (n = 2) are shown. ^∗∗∗Mann-Whitney p value < 0.0001.

(I) Focal adhesion size, based on p-paxillin staining, of 1,590 focal adhesions from siNT- and >5,000 from siCFL1-treated cells was quantified in ImageJ. Circles represent all individual measurements, and bars present mean ± SD. ^∗∗∗Mann-Whitney p value < 0.0001.

(J) IF staining of siNT-/siCFL1-treated cells for α-actinin and β-actin. Two examples of siCFL1-treated cells are shown, and zoomed images of boxed region are shown on lower right panels.

The scale bars represent 20 μm.