Fig. 4: PREP1 depletion affects the focal adhesion pathway.

a Representative confocal images showing adhesion foci (vinculin, red) and actin stress fibers (Phalloidin, green) in control (siLUC) and PREP1 depleted (siPREP1) U2OS cells. Nuclei are shown in blue (DAPI). Scale bar is 20 µm. b Box and whisker plot show quantification of focal adhesion area in control (siLUC) and PREP1 depleted (siPREP1) U2OS cells. Graph is derived from four independent experiments. N = 572 cells in siLUC and 611 cells in siPREP1. c Representative traction force heat-map images of control (siLUC) and PREP1 depleted (siPREP1) U2OS cells. The box plot shows mean traction force in kPa experienced by siLUC or siPREP1 U2OS cells. The data is derived from three independent experiments. N = 25 cells each in siLUC and siPREP1 cells. d Confocal images of vinculin foci and its corresponding binary image in U2OS cells expressing EGFP-KASH2ext or EGFP-KASH2. Box plot depicts the quantification of focal adhesion area in EGFP-KASH2ext or EGFP-KASH expressing U2OS cells. Data is derived from two independent experiments. N = 101 cells in EGFP-KASH2 and 120 cells in EGFP-KASH2ext. e Confocal images of vinculin foci and its corresponding binary image in control (siLUC) and SUN2 depleted (siSUN2) U2OS cells. Box plot shows focal adhesion area in the same cells. Data is derived from three independent experiments. N = 433 cells in siLUC and 628 cells in siSUN2. f Representative immunoblot showing the phosphorylation status of p130CAS, Src and FAK proteins in whole cell lysates of control (siLUC) and PREP1 depleted (siPREP1) U2OS cells. Corresponding total protein levels and GAPDH serve as loading control. Blot is representative of three independent experiments. Bar graph shows the relative protein expression in PREP1 depleted cells normalized to control cells across experiments. Error bars denote mean ± SE.