Fig 2: The effect of TLK2 silencing in ER+ breast cancer cells and benign breast epithelial cells.(a, b) TLK2 knockdown (KD) by esiRNA and siRNA transfection. (a) Cell proliferation was assessed by MTT assay in TLK2-high or low breast cancer cell lines or benign breast epithelial cells after TLK2 knockdown. Unt, untreated. Ctrl, control. Error bars represent the s.d. of three replicate measurements per condition. (b) Cell migration assessed by Boyden chamber transwell assay following TLK2 KD for 48 h in MCF7 and MDAMB361 cells using TLK2 esiRNA or siRNA. NIH 3T3 cells are used as chemo-attractant for MCF7 and MDAMB361 cells. Error bars represent the s.d. of three replicates measurements per condition. (c) Clonogenic assay was performed following Dox-inducible shRNA silencing of TLK2 in breast cancer cells. 0.5 mg ml−1 of Dox was used for this assay. Dox, doxycycline. Error bars represent the s.d. of three replicate measurements per condition. (d) TLK2 inhibition suppresses anchorage-independent growth of MCF7 and MDAMB361 cells. Dox (0.5 μg ml−1) was administered for 2 days to induce TLK2 shRNA and then soft-agar colony formation assay was performed. Error bars represent the s.d. of three replicate measurements per condition. (e) The cell growth inhibition after TLK2 KD using a siRNA#2 (with the same targeting sequence as the shTLK2) can be rescued by inducible TLK2 overexpression. Multiple silent mutations at the shTLK2 targeting region are introduced into the TLK2 ORF without affecting the amino acid sequence, to reduce the inhibition of ectopically expressed TLK2 by siRNA#2. TLK2 expression was induced by treating MCF7 cells with 100 or 2,000 ng ml−1 Dox; siTLK2 was then transfected and incubated for 2 weeks for clonogenic assay. Error bars represent the s.d. of two replicate measurements per condition. P values were calculated based on t-test. *P<0.05; **P<0.01; ***P<0.001. Dox, doxycycline.

Fig 3: TLK2 inhibition results in impaired G1/S progression and induction of apoptosis in TLK2-amplified luminal breast cancer cells.(a) Flow cytometry results showing cell cycle changes after TLK2 knockdown in asynchronized MCF7 and MDAMB361 cells. 10 nM siRNAs (for MCF7 cells) or indicated concentration of siRNAs (for MDAMB361 cells) were transfected for 72 h. Ctrl, Control. (b) Cell apoptosis assessed by Annexin V assay in asynchronized MCF7 and MDAMB361 cells following TLK2 knockdown via 20 nM esiTLK2 or siTLK2#1 transfection for 72 h. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t-test. *P<0.05; **P<0.01; ***P<0.001. (c) Cell cycle profile of synchronized MCF7 cells (by double thymidine block) after TLK2 inhibition. After TLK2 knockdown for 24 h, MCF7 cells were synchronized at the G1/S border using 2.5 mM double thymidine (DT), and then released. Cells were collected every 2 h after cell cycle release for up to 12 h, and analysed for DNA content using flow cytometry. (d) Western blot was done to examine the changes of key signalling molecules involved in G1/S cell cycle regulation using the cell lysates obtained from the same experiment as in 7c. ‘DT' indicates synchronized MCF7 cells by DT block. (e) A schematic of normal G1/S cell cycle signalling and alternations following TLK2 inhibition (black arrows). In normal cell cycle, the cyclin E level starts to increase in late G1 phase, and then collapses as the cells enter S phase53, followed by increased cyclin A expression5455. Rb regulates G1/S transition by repressing the E2F transcription factors that control the expression of cyclin A. Once Rb is phosphorylated (that is, at S807/S811), it releases E2Fs, which will allow cells to enter S phase5657. p27 inhibits the two G1 cyclin/cdk complexes, cyclin D/Cdk4 and cyclin E/Cdk2 (refs 36, 40), both of which are the key upstream kinases of Rb (ref. 37). During normal G1/S progression, the p27 proteins complexed with G1 cyclin/cdks were phosphorylated by the p27-free cyclin E/Cdk2 complexes at T187, which were then targeted for SKP2-mediated proteasome degradation58. D, Cyclin D. E, Cyclin E. A, Cyclin A. R, restriction point.

Fig 4: The phenotypic changes after inducible ectopic expression of TLK2 in the MCF10A benign breast epithelial cells.(a) Bar chart showing TLK2 gene expression in a panel of breast cancer and benign cell lines based on exon expression array data21. (b) Western blot detecting the TLK2 proteins ectopically expressed in MCF10A cells after induction with different doses of doxycycline (Dox). (c) Cell survival following induction of TLK2 expression in MCF10A cells was measured by clonogenic assay. Error bars represent the s.d. of three replicate measurements per condition. (d) Transwell migration and matrigel invasion assays. After 2-week induction of TLK2 expression with different doses of Dox, Dox was either continued or withdrawn to test if the increased cell migration and invasion is dependent on TLK2 expression. Error bars represent the s.d. of three replicate measurements per condition.

Fig 5: 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.