Figure 2.

ARNO regulates SFs migration and assembly of focal adhesions. (A) SFs were seeded in migration chambers and grown until monolayer confluence. Cells were then treated with either Allstars control or ARNO siRNAs, when inserts were removed to perform migration assays. Pictures show one representative experiment, superimposed black lines delineate the cell-free area. Bar chart shows the mean of cell migration distance ± SEM from three independent experiments calculated with ImageJ software. (B) SFs were labelled with proliferation dye eFluor 670 (eBioscience), analysed by flow cytometry (day 0) or treated with control or ARNO siRNA, when cells were maintained in culture. After 5 days, mean fluorescence intensity was evaluated in live cells (identified by low DAPI staining). Histogram shows one representative experiment. Mean fluorescence intensity (MFI) of three independent experiments is shown in the column graph, error bars represent ± SEM. (C) Representative immunofluorescence staining of Vinculin (red) and F-actin (green) in SFs treated with Allstars control or ARNO siRNA. Scale bar: 50um. (D) Number, length and area of vinculin positive areas were analysed with ImageJ in SFs treated with Allstars control or ARNO siRNA. Each dot represents one single cell, data are from one representative experiment. (E) Mean of number, length and area of vinculin positive areas from three independent experiments, in SFs treated with Allstars control or ARNO siRNA. Error bars error bars represent ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann-Whitney test.