Fig. 4

LCK inhibitors strongly reduces SAS invasiveness. A, B 2D cell layers of highly invasive SAS subclones (H, S) and parental SAS were scratched and simultaneously treated with LCK inhibitors, i.e. LCKi (500 nM) or dasatinib (100 nM). Representative pictures were taken 8 h after the scratch (scale bar = 400 μm). The initial scratch wound is labeled in purple and the cell layer in orange. The normalized slope of a linear regression of the wound density over time is plotted (n = 3). C, D LCKi and dasatinib treated 3D spheroids were embedded in matrigel to analyze tumor cell invasion. Pictures were taken after 48 h (n = 3). The invasive front is marked in turquoise (scale bar = 400 μm). E, F 3D spheroid growth of SAS subclones and parental SAS cells upon treatment with LCKi or dasatinib for 48 h. Measured area was used to compute the volume increase. Representative pictures are shown (n = 3), scale bar = 200 μm. G Western blot after LCKi and dasatinib treatment. Total Paxillin and Paxillin phosphorylation at tyr118 was detected. Rpl7 served as loading control. H Relative amount of tyr118 phosphorylated Paxillin after inhibitor treatment. Quantification was done by calculating the ratio of phosphorylated and total Paxillin in each sample and normalizing these values to the DMSO control treatment (n = 3; two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001)