We extended our observations by testing whether expressing the 602F-EPS8 mutant was sufficient to alter the ability of HN4 cells to migrate. Moreover, Y602F- and FFFF-EPS8 mutants reduced mitogenesis and motility. Strikingly though, Forsythoside A FFFF- or Y602F-EPS8 mutants actually promoted tumorigenicity compared with control cells. Conclusions Phosphorylation of EPS8 at Y602 is crucial for signalling to the cell cycle and may provide insight to explain reduced efficacy of dasatinib treatment. is the tumour volume, is the tumour width and is the tumour length. Tumour tissues were sectioned, fixed, stained with haematoxylin and eosin and examined under a light microscope. Immunohistochemistry Detection of EPS8 in mouse tumour sections was carried out as follows. Five-micrometre tissue sections were dewaxed in Safe-Clear (Fisher Scientific, Pittsburgh, PA) and rehydrated, and antigen retrieval was performed by incubation in Retrievagen A (BD Biosciences, CA) at 95?C for 30?min. After cooling to ambient temperature, endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide for 15?min; slides were washed in TBS pH 8.0 and then blocked using a commercially available kit (M.O.M. Immunodetection KitPeroxidase; Vector Laboratories, Burlingame, CA). Sections were then incubated with EPS8 antibody (12.5 g/ml) or the equivalent concentration of normal mouse IgG as control at ambient temperature for 1?h, washed in TBS and then incubated sequentially with biotinylated secondary antibody and streptavidin peroxidase reagent, developed using 3, 3-diaminobenzidine substrate, counterstained with haematoxylin, dehydrated, mounted and imaged by brightfield microscopy (Keyence Corporation, Itasca, IL). RNA sequencing For whole-transcriptome analysis, cells were cultured to 70% Forsythoside A confluence under standard culture conditions. Alternatively, HN4 cells were treated with 400?nM dasatinib or equivalent volume of the vehicle (DMSO) as a control for 24?h. Triplicate cultures were lysed individually in Trizol, total RNA was prepared and then library construction, sequencing and bioinformatics analyses were carried out by a commercial source (Novogene, Sacramento, CA). Statistical analysis Students test or one-way analysis of variance with post multiple comparisons were performed using the GraphPad Prism software to analyse the data obtained from western blotting, migration, proliferation, immunostaining and tumorigenicity experiments. values 0.05 were considered to be statistically significant. Results Blocking tyrosine phosphorylation at the Src target sites in EPS8 reduces Forsythoside A the expression of cell cycle regulators Src activity has been previously found to be involved in modulating cell signalling downstream of EGFR and participates in the regulation of cell proliferation.42,43 Moreover, EPS8 overexpression leads to enhanced cell proliferation and increased expression of multiple cell cycle regulators.33 Previous studies have documented constitutive phosphorylation of EPS8 in some cancer cell lines.24 Therefore, Forsythoside A we determined the phosphorylation status of EPS8 in HN4 cells. As shown in Supplementary Fig.?S1A, B, EPS8 is tyrosine phosphorylated in HN4 cells in the absence of growth factors. As EPS8 is a Src substrate,30 we sought to determine the impact of blocking Src phosphorylation of EPS8 on the expression of cell cycle mediators. Thus HN4 cells were first stably transfected with FFFF-EPS8 mutants, total cell lysates were prepared and the expression of FOXM1 and two of its targets, AURKA and AURKB, was determined by western blotting. As indicated in Fig.?1aCd, blocking EPS8 phosphorylation at all four sites (FFFF-EPS8 mutant) significantly reduced the expression Forsythoside A of FOXM1, AURKA and AURKB compared with the non-transfected control, showing the importance of Src phosphorylation sites of TFR2 EPS8 in controlling the expression of these cell cycle regulators. To determine whether blocking Src-mediated EPS8 phosphorylation is responsible for the reduced expression of cell cycle regulators, HN4 cells were treated with 400?nM of the Src inhibitor, dasatinib, for 24?h. Total cell lysates were prepared and the effect on cell cycle regulators was determined by western blotting. As shown in Fig.?1eCh, there was a significant reduction in the expression of FOXM1, AURKA and AURKB with dasatinib treatment relative to vehicle-treated controls. Furthermore, phosphorylation of EPS8 on tyrosine is reduced, but not completely abolished, by treatment with dasatinib (Supplementary.