Fig 2: Inhibition of Notch 1 expression with Notch 1-specific-siRNA suppressed the activity of the Akt/mTOR signaling pathway in clear cell renal cell carcinoma 786-O cells. Expression of key components in the Akt/mTOR signaling pathway was evaluated by western blot analysis. (A) Inhibition of Notch 1 expression had no effect on total Akt expression, but inhibited the expression of p-Akt, p-mTOR and p-P70S6K in a dose-dependent manner. Quantitative determination of (B) Akt and p-Akt, and (C) p-mTOR and p-P70S6K expression following normalization by ß-actin. Data are presented as the mean ± standard deviation of three independent western blot assays and presented as column diagrams. *P<0.05, **P<0.01 vs. control. siRNA, small interfering RNA; mTOR, mechanistic target of rapamycin; p-, phosphorylated-.

Fig 3: 108600 suppresses the growth of TNBC stem cells.A Chemical structure of 108600. B IC50 values for inhibition of 108600 target kinases. C 108600 inhibits phosphorylation of CK2a, DYRK1A and TNIK substrates. Lysates derived from MDA-MB-231 and Hs578T cells treated for 24 h with 108600 at the indicated concentrations were probed for expression of phospho-AKT1 (Ser129), phospho-CYCLIN D1 (Thr286), and AXIN2. The blots are representative of three independent experiments. Samples derive from the same experiment and the blots processed in parallel. D GI50 values for growth inhibition of breast cancer and normal cells treated with 108600. E Simultaneous siRNA-mediated knockdown of CK2a, DYRK1A, and TNIK suppresses proliferation of MDA-MB-231 and Hs578T cells. siRNA-transfected cells were counted on the indicated days. All data represent mean values ± standard deviation obtained from three independent biological replicates. The statistical difference between the target and nontarget siRNA treated groups was tested using two-way ANOVA (two-sided). Knockdown was confirmed by western blot analysis, which was performed three times. Samples derive from the same experiment and the blots processed in parallel. F 108600 treatment suppresses colony and G mammosphere formation of CD44high/CD24low stem cell fraction of MDA-MB-231 and Hs578T cells. For colony assays, CD44high/CD24low cells were purified by FACS, treated with the indicated concentrations of 108600 for 72 h, washed and allowed to grow for 7–14 days in drug-free medium. Cells were fixed with 4% paraformaldehyde in PBS, stained with crystal violet solution and colonies quantified microscopically. All data points represent mean value ± standard deviation. Scale bars = 50 µm. Values in F and G represent the mean percentage of colony and sphere formation ±SD, respectively, relative to that of DMSO, which was set to 100%, in three independent experiments. Source data are provided as a source data file.

Fig 4: Potential BRAFi resistance candidates. Blocking of CD13/ANPEP leads to dephosphorylation of EPHA2 on S897 and increased apoptosis. (a) Protein expression levels of the potential resistance candidate protein CD13/ANPEP analyzed by immunoblotting in presence of 1 µM of vemurafenib or DMSO as control treatment for 24 h in parental A375 and three BRAFi-resistant sublines. ß-actin was used as control. (b) Immunofluoroscence showing CD13 expression in A375, A375VR4 and SKMEL24 cells. CD13 is stained with Alexa Fluor 594 (red) and nucleus is counterstained with DAPI (blue). (c) EPHA2, AKT1 and RSK phosphorylation was decreased after 48 h treatment with anti-CD13 blocking antibody. (d) Induction of apoptosis after 48 h treatment with anti-CD13 blocking antibody in A375, A375VR4 and SKMEL24 cells. Apoptosis was measured by flow cytometry based annexin V-PI staining. (Error bars represent S.D. of the mean of two independent experiments each in three replicates)