Fig. 5

The amplification-associated transcription coupling (ATC) locus TLK2 is an actionable target for 17q23-amplified breast cancers. a. DNA copy-numbers of TLK2 across normal, benign and breast cancer cell lines. Quantitative PCR assays were used to determine copy number alterations. Data represent mean ± standard error of the mean (SEM) of three independent experiments. b. Western blot detecting TLK2 protein expressed in normal, benign and breast cancer cell lines. Loading amount was normalized with α-tubulin. Data represent mean ± standard error of the mean (SEM) of three independent experiments. c. Antiproliferative effects of antipsychotics on breast cancer cell lines. Cells were treated with or without phenothiazine derivatives (PPH, TFP and TRD) at various concentrations. Phase confluence percentage of the cells was collected every 12 h totally for a period of 5 days using IncuCyte ZOOM live-cell imaging system. Quadruplicate replicates were used in each experiment. Quantitative analysis of cell growth 120 h after drug treatment by one-way ANOVA (vs Ctrl). d. DNA damage response in cells treated with or without PPH (5.0 μM) was analyzed with Western blot and immunostaining. Double strand breaks were induced by bleomycin. γH2AX was accumulated more in MCF-7 and MDA-MB-157 cells treated with PPH. No significant difference of RAD51 expression was noted between the cells treated with or without PPH. Representative immunofluorescence images of γH2AX foci (green) were shown in MCF-7 cells treated with or without PPH (right). Scale bar, 30 μm. e. Western blot analysis of TLK2 siRNA knockdown efficiency in MCF-7 cells. TLK2 protein level was markedly decreased in the cells treated with TLK2 siRNA. f. Inhibitory effect of TLK2 knockdown and PPH treatment (5.0 μM) on cell proliferation of MCF-7 and MDA-MB-231 cells. Phase confluence percentage of the cells was collected every 12 h totally for a period of 5 days using IncuCyte ZOOM live-cell imaging system. Quantitative analysis of cell growth 120 h after drug treatment by one-way ANOVA (vs siCtrl). g. Western blot analysis of the indicated signaling molecules in MCF-7 cells treated with bleomycin, TLK2 siRNA knockdown and/or PPH