Fig. 3

Knockdown of LCK inhibits SAS cell migration in vitro. A LCK mRNA abundance was measured via RT-qPCR in primary OSCC samples (n = 117) and metastasis (n = 12), and B four HNSCC-derived cells (n = 3). Normalization was performed using RPLP0 and POLR2A (A), or PPIA (B) and the median of the primary tumor values or the average expression across all cell lines, respectively. C Detection of LCK protein in HNSCC-derived cell lines and D its quantification relative to Rpl7 and the mean level across all cell lines. Significance test was performed using student’s t-test comparing primary tumor samples versus metastasis (A), one cell line with the other three (B, D). E LCK mRNA abundance as measured via RT-qPCR in twenty SAS subclones. Relative Expression normalized to RPLP0 and PPIA as well as the average of all clones. High invasive clones are marked in red and low invasive clones in green. One-way ANOVA comparing each subclone with the subclones of the second and third quartile was used to determine significance (n = 3). F Correlation analysis of LCK mRNA abundance in each clone and the respective clonal invasion factor of SAS subclones grouped by Vimentin and N-cadherin level. G Representative Western blot of LCK upon its depletion in clones H, S and SAS. Vinculin and Rpl7 served as loading controls. H Quantification of LCK protein abundance in highly invasive clones (H & S) and SAS parental cells upon siRNA-mediated depletion (n = 3). I Analysis of 2D migration upon LCK depletion. Representative pictures of the scratch wound assay were taken 4 h after the scratch (scale bar = 400 μm). The initial scratch wound is labeled in purple and the cell layer in orange. J The normalized slope of a linear regression of the wound density over time is plotted (n = 5). Two-way ANOVA was used for statistical testing (H, J). (*p < 0.05, **p < 0.01, ***p < 0.001)