Figure 4. 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.