Figure 3: The phenotypic and cell signalling changes after ectopic expression of TLK2 in the T47D ER+/Her2− luminal breast cancer cells.

(a) Western blot detecting TLK2 protein ectopically expressed in T47D cells after induction with different doses of Dox. (b) Cell survival following induction of TLK2 expression in T47D cells was measured by clonogenic assay. Error bars represent the s.d. of three replicate measurements per condition. (c) TLK2 overexpression significantly enhanced anchorage-independent growth of T47D cells. TLK2 was overexpressed in T47D by treating with 100 ng ml⁻¹ Dox before the soft-agar assays were performed. Error bars represent the s.d. of three replicate measurements per condition. (d) Transwell migration and matrigel invasion assays. Following TLK2 induction for 2 weeks, Dox was either continued or withdrawn to test if the increased cell migration and invasion is dependent on TLK2 overexpression. Error bars represent the s.d. of two replicate measurements per condition. (e) Alterations of key signalling molecules in breast cancer were examined by Western blot following TLK2 overexpression in T47D. TLK2 was induced by 200 ng ml⁻¹ Dox for 2 weeks. Dox, doxycyclin. (f) Transwell migration assays following SRC, EGFR or FAK knockdown in T47D cells overexpressing TLK2. The indicated concentration of siRNAs against SRC or 20 nM of siRNAs against EGFR or FAK were transfected for 3 days following induction of TLK2 expression in T47D cells for two weeks (200 ng ml⁻¹ Dox). Western blot validation of SRC, EGFR or FAK silencing in T47D cells overexpressing TLK2 was shown in Supplementary Fig. 5. Error bars represent the s.d. of two replicate measurements per condition. (g) Co-immunoprecipitation of TLK2 with SRC in engineered MCF7 cells inducibly expressing Flag-tagged TLK2. Engineered MCF7 cells was treated with 200 ng ml⁻¹ Dox for 48 h and nuclear or cytoplasmic proteins were purified following subcellular fractionation. IP was performed using an established monoclonal antibody against SRC after conjugation with agarose beads. Western blot was performed using an anti-Flag antibody to detect the presence of TLK2 in SRC complex. Dox, Doxycyclin.