Fig. 3: AXL localizes at focal adhesion (FA) sites and modulates their turnover.

a AXL localizes at FA sites. Representative confocal images of MDA-MB-231 cells treated either with DMSO or 1 μM R428. Proximity ligation assay (PLA) was used to analyze localization of AXL at PXN-positive FAs. Scale bar, 20 μm. b Quantifications of the number of PLA puncta per cell per condition. ****P < 0.0001. (n = 3 experiments, 15 cells per condition per experiment). c, d AXL modulates the number of FAs per cell. Representative confocal images of Hs578T cells treated with DMSO or 1 μM R428, transfected with 100 nM of siCTRL or siAXL (c), serum starved and treated with GAS6 for 20 min, or serum starved and treated with GAS6 and R428 (d). Cells were stained for PXN (green) and Phalloidin (magenta). Scale bar, 10 μm. e Quantification of the FA number per cell depicted in c, d. ***P = 0.0001, ****P < 0.0001 (n = 3 experiments with 30 cells per condition). f MDA-MB-231 GFP-PXN-expressing cells treated with DMSO or 1 μM R428 were imaged live by spinning disk microscopy for a period of 30 min to assess the dynamics of FA turnover. Magenta arrow heads point toward a FA that was followed for its assembly and disassembly times. Scale bar, 5 μm. g, h Quantification of FA lifetime (g) and their assembly and disassembly times (h) of cells depicted in f and MDA-MB-231 GFP-PXN-expressing cells treated with GAS6 for 30 min or transfected with 100 nM siAXL and imaged as mentioned in f. ***P < 0.0001 (g), **P = 0.00899 (g), **P = 0.000014 (h), ***P = 0.00009 (h), ****P = 0.000009 (h) (n = 3 experiments with 90 FAs followed per condition). Data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value, and the lower whisker extends from the hinge to the smallest value (b, e, g, h). Two-tailed unpaired t test was used. Source data are provided as a Source data file.