Fig. 4

ZL170 inhibits Snail and Slug expression, reverses the EMT program and reduces EMT-dependent FLP formation in TNBC cells. a Representative immunoblot analyses of Snail and Slug levels in MDA-MB-231 and PyMT cells that were treated with vehicle or ZL170 at 20 μM for different times (n = 3). b and c Immunoblot analyses of Snail and Slug levels in the indicated cells that were treated with vehicle or ZL170 at 5, 10 and 20 μM for 3 h (b) or in the indicated cells that were treated with vehicle, ZL170 or LY2157299 at 20 μM for 1 h and 3 h (c). n = 3 independent experiments. d-g ZL170 efficiently abolished TGFβ1-stimulated and BMP4-stimulated expression of Snail and Slug in MDA-MB-231 cells as indicated by qPCR (d), immunoblot (e) and immunofluorescent (f and g) analyses (n = 3). Cells were treated with TGFβ1 or BMP4 in combination with ZL170 at 20 μM for 3 h. Data are represented as mean ± S.D. ** P < 0.01, one-way ANOVA test. h and i ZL170 efficiently reduced Snail and Slug levels in TGFBR1-T204D stably expressing cells as indicated by representative immunoblot (h) and qPCR (i) analyses (n = 3). Cells were treated with the compound for 3 h. j and k Representative immunoblot analyses of fibronectin, N-cadherin, vimentin or E-cadherin levels in MDA-MB-231 (j) and 4 T1 (k) cells that were treated with vehicle or ZL170 at 5, 10 and 20 μM for 48 h (n = 3). (L and M) Immunofluorescent staining of fibronectin (l) and E-cadherin (m) in the indicated cells that were treated as described in J and K, respectively (n = 3). n Immunofluorescent staining of E-cadherin in the indicated cells that were treated with TGFβ1 or BMP4 in combination with ZL170 at 20 μM for 48 h (n = 3). o Immunofluorescent staining of F-actin and cortactin in vehicle- and ZL170-treated MDA-MB-231 cells. Nuclear, DAPI (blue). Arrows denote F-actin⁺/cortactin⁺ FLPs (n = 3). Scale bars = 20 μm (f, g, l, m, n) and 5 μm (o)