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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Discovery of a natural small-molecule compound that suppresses tumor EMT, stemness and metastasis by inhibiting TGFβ/BMP signaling in triple-negative breast cancer

Fig. 3

ZL170 is a dual inhibitor of TGFβ and BMP kinase receptors and reduces activation of Smads in TNBC cells. a In vitro kinase activity assays of the inhibitory efficacy of ZL170 on phosphorylation of the substrates (BMPR1A, BMPR1B, BMPR2, TGFBR1, ACVR1B, ACVR1 and TGFBR2). b Molecular docking analysis of the potential binding between ZL170 and TGFβ/BMP receptors. Illustration of surface crystal structure of ZL170 against BMPR1A, BMPR1B, BMPR2 and TGFBR1 shown. c Representative immunoblot analyses of the levels of phospho-Smad1/5 and phospho-Smad2/3 (and their total forms) in MDA-MB-231 and PyMT cells that were treated with vehicle or ZL170 at 20 μM for different times (n = 3). d Immunoblot analyses of the levels of phospho-Smad1/5 and phospho-Smad2/3 (and their total forms) in the indicated cells that were treated with vehicle or ZL170 at 5, 10 and 20 μM for 3 h. n = 3 independent experiments. e ZL170 efficiently abolished TGFβ1-stimulated and BMP4-stimulated expression of phospho-Smads in MDA-MB-231 cells as indicated by representative immunoblot analyses (n = 3). Cells were treated with TGFβ1 or BMP4 in combination with ZL170 at 20 μM for 3 h. f and g Representative immunofluorescent staining of phospho-Smads (f) and Smads (g) in resting, TGFβ1-stimulated and BMP4-stimulated MDA-MB-231 cells (n = 3). (H) ZL170 efficiently reduced the levels of phospho-Smads in TGFBR1-T204D (left panel) or BMPR1A-Q233D (right panel) stably expressing cells as indicated by representative immunoblot analyses (n = 3). Cells were treated with the compound for 3 h. i SBE promoter luciferase reporter assays in MDA-MB-231 cells that were treated with TGFβ1 and increasing doses of ZL170 (left panel) or in TGFBR1-T204D stably expressing cells treated with increasing doses of ZL170 (right panel) (n = 3). j BRE4 promoter luciferase reporter assays in MDA-MB-231 cells treated with increasing doses of ZL170 (left panel) or in BMPR1A-Q233D stably expressing cells treated with increasing doses of ZL170 (right panel). k-m Boyden chamber invasion assays of MDA-MB-231 cells stably expressing control-shRNA or TGFBR1-shRNA (k and l). Quantification of invaded cells were shown in (M). n = 3 independent experiments. n-p Boyden chamber invasion assays of MDA-MB-231 cells stably expressing control-shRNA or BMPR1A-shRNA (n and o). Quantification of invaded cells were shown in (p). n = 3 independent experiments. Data are represented as mean ± S.D. * P < 0.05, ** P < 0.01, one-way ANOVA test. Scale bars = 20 μm (f, g) and 200 μm (l, o)

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