Figure 2.

Inhibition of the BMP pathway strongly impairs the SNAIL1-induced EMT in colorectal cancer cells. (a) Schematic depiction of the BMP signaling pathway. The two inhibitors Noggin and LDN193189 interfere with signal transduction by sequestering BMP ligands and inhibiting BMP type I receptor A (ALK3), respectively. (b) Western blot analyses of whole-cell lysates. Names of detected proteins are indicated on the right. Cells were left uninduced or were treated with 0.1 µg·mL⁻¹ Dox and 50 nM LDN193189 (L), or DMSO (D) for 72 h. Positions of molecular weight (M_W) standards in kDa are given on the left. Detection of ACTIN was used as control for equal loading. (c) Western Blot analyses of whole-cell lysates. Names of detected proteins are indicated on the right. Cells were left uninduced or were treated with 0.1 µg·mL⁻¹ Dox and 100 ng·mL⁻¹ Noggin for the indicated time spans. Positions of molecular weight (M_W) standards in kDa are given on the left. Detection of ACTIN was used as control for equal loading. (d) qRT-PCR analyses of mRNA expression in LS174T-Snail1-HA cells. Where indicated, cells were treated with 0.1 µg·mL⁻¹ Dox, 50 nM LDN193189 (L), DMSO (D), or 100 ng·mL⁻¹ Noggin (N) for 72 h. Shown is the mean+SEM; n = 3. Rel. expr.: relative expression normalized to that of GAPDH. ns: not significant. *: p < 0.05, **: p < 0.01. (e) Representative phase contrast images of LS174T-Snail1-HA cells treated with 0.1 µg·mL⁻¹ Dox and DMSO, 50 nM LDN193189 (LDN), or 100 ng·mL⁻¹ Noggin (NOG) for 72 h as indicated. Scale bar: 100 µm. (f) Spheroid invasion assay of LS174T-Snail1-HA cells treated with 0.1 µg·mL⁻¹ Dox and DMSO, 50 nM LDN193189 (LDN), or 100 ng·mL⁻¹ Noggin (NOG) for 96 h as indicated. Two representative spheroids are shown for each condition. Scale bar: 200 µm.