Fig 2: PTEN protein phosphatase activity is sufficient to inhibit activation of PTK6. a A graphical representation of the PTEN domain structure. Mutations in the phosphatase and tail domains including C124S (phosphatase-dead), G129R (phosphatase-deficient), A4 (tail mutant), Y138L (protein phosphatase-dead), G129E (lipid-phosphatase dead), Y138L A4 (protein phosphatase-dead, tail mutant), C124S A4 (phosphatase-dead, tail mutant), or G129R A4 (phosphatase-deficient, tail mutant) are shown. b PC3 cells stably expressing empty vector, Palm-WT, or Palm-YF were transiently transfected with empty vector, wild-type PTEN, or recombinant PTEN mutants. Changes in phosphorylation on PTK6 Y342 and Y447 were monitored by immunoblotting. c PTEN was immunoprecipitated from PC3 cells stably expressing PTK6-WT and constitutively-active PTK6-YF which were transiently transfected with empty vector, wild-type PTEN, or PTEN mutants G129E, Y138L, or PTEN A4. Bound PTK6 protein was subjected to SDS–PAGE and observed by western blot d PC3 cells stably expressing PTK6-WT and constitutively-active PTK6-YF which were transiently transfected with empty vector, wild-type PTEN, or PTEN mutants G129E, Y138L, or PTEN A4. Cells were then probed for PTK6 PY342 (green). Cells were counterstained with DAPI (blue). Scale bar, 10 µm. e Purified PTEN, PTP1B, SHP1, and the cytoplasmic catalytic domain of CD45 proteins were used in a phosphatase assay using PTK6 as a substrate. The reaction was run for the described time interval. Changes in PTK6 phosphorylation at Y342 and Y447 were detected by immunoblotting

Fig 3: SRC loss does not impact activation of AKT, FAK, and EGFR by PTK6.A, total cell lysates of PC-3 cells that stably express empty vector or membrane targeted-constitutively active PTK6 (Palm-YF) were harvested for immunoblotting (Top). Phospho-protein signals were quantified and normalized to total proteins, and two tailed Student's t-test was used for statistical analysis (Bottom), **p < 0.01, ***p < 0.001. B, siRNA was used to knock down SRC gene expression in PC-3 cells stably expressing empty vector or membrane-targeted constitutively active PTK6 (Palm-YF). PC-3 cells were transfected with SRC siRNA (siSRC) or control siRNA (siCtrl) and harvested after 48 h. Total cell lysates were subjected to immunoblotting.

Fig 4: PTK6 hypomethylation induces OSCC carcinogenesis by increasing PTK6 transcriptional expression in the animal model. (a) Flowchart of exposure of mice to carcinogens (200 µg/mL of 4-NQO and 500 µg/mL of arecoline) in drinking water. The mice treated with 4-NQO and arecoline and corresponding control mice were sacrificed at 8, 16, 20, and 29 weeks for analysis. (b) Methylation analysis of PTK6 in the tongue tissues of OSCC mice at different time points. (c) PTK6 transcriptional expression in the tongue tissues of the OSCC mice at different time points. (d) The protein expression of PTK6 in the tongue tissues of the OSCC mice at different time points. * p < 0.05. ** p < 0.01; *** p < 0.005.

Fig 5: Downregulation of PTK6 suppressed OSCC cell migration and invasion. (a) OCMC and OECM1 cell lines were used to knock down PTK6 by using shRNA. The mRNA and protein expression were examined through real-time PCR and Western blotting in PTK6 knockdown cells. (b) Cell viability of control and PTK6 knockdown cells was evaluated using a WST-1 assay. Absorbance was measured at 450 nm at seven time points (0, 24, 48, 72, 96, 120, and 144 h) to calculate the proliferation rate. (c) The migration abilities of control and PTK6 knockdown cells were examined using a transwell assay. Migrated cells were quantified using ImageJ. (d) The invasive abilities of control and PTK6 knockdown cells were measured using a transwell Matrigel assay. Invasive cells were quantified using ImageJ. ns means not significant. **** p < 0.001.