Fig. 5

Cellular defects by PINCH-1 4A mutation. a Overexpression experiment. HEL cells expressing EGFP-PINCH-1 WT or -1 4A cells were stimulated with 800 nM PMA for 10 min, spread on 20 μg ml⁻¹ fibrinogen for 1 h, fixed and stained with Alexa 647-phalloidin to visualize actin and anti-vinculin antibodies to mark FAs. Transfected cells were identified by EGFP fluorescence. Top panels. WT EGFP-PINCH-1 promotes the formation of F-actin stress fibers. By contrast, the stress fibers were substantially disrupted in the cells expressing the mutant PINCH-1-4A (bottom panels). Scale bar, 10 µm. b Quantitative analysis of inhibition of cell spreading by PINCH-4A as compared with the WT PINCH-1. Cells were spread on fibrinogen as described in (a). The areas of EGFP-positive cells were measured using ImageJ software. *P ≤ 0.001. In total, 150 cells were quantified in each sample. c CRISPR/Cas9-based knock-in mutation experiment. HeLa clone (2F5) with frame-shifted PINCH deletion mutant, PINCH-∆C (2F5) was transfected by pEGFP vector (V), pEGFP-PINCH wildtype (WT) or pEGFP-PINCH mutant (4A) and sorted for 2 h spreading on fibronectin coated coverslips (10 µg cm⁻²). Focal adhesions and stress fibers are clearly disrupted by PINCH 4A mutation. Cells were fixed and stained for GFP, ILK, and F-actin. Images were then captured with confocal microscope, under 63x magnification. Scale bar, 20 µm. d Quantification of cell spreading with physiological level of PINCH WT/4A expression (P1) in HeLa clone (2F5). Spreading area of 44 cells from each group were quantified with Image Pro plus software based on F-actin staining. Significance was calculated by t test. **P < 0.01. The box of the boxplot illustrated the upper and lower quartile for each population (EGFP vector, EGFP-PINCH1-WT and EGFP-PINCH1-4A). Median of spreading area is marked by a horizontal line within the box. The attached whisker indicates the range, and the discrete points (•) are the outliers