Fig 1: Effects of TTK inhibition on TGF-β signaling and miR-21 expression in MDA-MB-231 breast cancer cells. a Comparison of the SB505124 TGF-β inhibitor and the NMS-P715 TTK inhibitor for their effects on Smad3 phosphorylation in MDA-MB-231 cells, as measured by western blotting. b Detection of EMT markers E-cadherin and ZO1 in and images of MCF10A mammary epithelial cells following treatments with TGF-B, the NMS-P715 TTK inhibitor, and their combination. c–e Detection of mir-21 expression by real-time PCR in MDA-MB-231 cells with the knockdown of TTK or TTK/KLF5 by siRNA (c), treatment with the NMS-P715 TTK inhibitor (d), and lentivirus mediated KLF5 overexpression (e). *p < .05, **p < .01. f Protein expression of EMT markers in MDA-MB-231 cells with siRNA knockdown of TTK and miR-21 overexpression. g, h Representative images of invaded cells (g) and their quantification in motility and invasion assays. **p < .01
Fig 2: TTK inhibition upregulates the expression of miR-200a and miR-200c to attenuate EMT in MDA-MB-231 cells. a, b Detection of miR-200a and miR-200c expression by real-time PCR in MDA-MB-231 cells treated with the NMS-P715 TTK inhibitor (a) or TTK and KLF5 siRNA (b, c). d, e Expression of EMT markers in MDA-MB-231 cells treated with NMS-P715 in combination with pools of miR-200 mimics. f, g Representative images of cells (f) and detection of mRNA expression of KLF5, CDH1, ZEB1, and VIM by real-time PCR (g) in MDA-MB-231 cells treated with NMS-P715 in the presence or absence of inhibitors against miR-200a or miR-200c. **p < .01; *p < .05
Fig 3: TTK inhibition attenuates the mesenchymal status of MDA-MB-231 and Hs578t TNBC cells. a Vimentin and E-cadherin protein expression in MDA-MB-231 cells stably expressing the pLKO.1 vector or the shTTK shRNA, as detected by western blotting. b Detection of vimentin expression by immunofluorescence staining in MDA-MB-231 cells stably expressing pLKO.1 or shTTK. c–h Detection of EMT markers in MDA-MB-231 (c–e, i) and Hs578t (f–h, j) TNBC cell lines treated with the NMS-P715 TTK inhibitor by western blotting for protein (c and f, upper), immunofluorescence staining for vimentin (d, g lower), and by real-time PCR for mRNA (e, h–j). *p < .05,**p < .01. Bright field (BF) cell morphology is also shown (d and g upper)
Fig 4: TTK inhibition suppresses the proliferation of TNBC cells. a, b Cell viability in MDA-MB-231 and Hs578t TNBC cells as determined by the SRB assay. **p < .01, c–e Representative colony formations and validation of stable TTK downregulation via viral delivery of shRNA in MDA-MB-231 cells. *p < .05, **p < .01. f Detection of TTK in a panel of cell lines
Fig 5: Effects of CFI-402257 and AICAR on the main target proteins in MDA-MB-231 cells. (A) Western blot analysis of p-TTK and TTK. Cells were pretreated with 50 ng/ml nocodazole for 17 h before treatment with CFI-402257 and AICAR and their combination for 4 h. (B) Western blot analysis of p-AMPK and AMPK. Cells were treated with CFI-402257 and AICAR and their combination for 24 h. (C) Representative histogram of protein expression levels. The values represent the mean ± SEM. ###P<0.001 vs. nocodazole group; **P<0.01 and ***P<0.001 vs. the control group. p-, phosphorylated.
from Cell Signaling Technology for TTK (D15B7) Rabbit mAb