Snai1

Whole cell lysates were prepared in RIPA buffer (Sigma) and quantified using Bradford Protein Assay (Bio-Rad). Twenty micrograms of lysates were resolved on Bolt^® 4–12% Bis-Tris Plus gels using Bolt^TM MOPS SDS Running buffer and transferred to a Hybond C super nitrocellulose membrane (GE Healthcare). After blocking to prevent non-specific binding in 5% milk in PBST for 1 h at room temperature, membranes were incubated with the specific primary antibodies overnight at 4 °C. The following primary antibodies were used: E-cadherin (Takara Bio Inc., M106, clone HECD-1, 1:1000 dilution), Occludin (Cell Signaling, #5446, 1:1000 dilution), Vimentin (Cell Signaling, #5741, 1:3000 dilution), ZEB1 (Santa Cruz, sc-25388, 1:500 dilution), SNAI1 (Santa Cruz, sc-28199, 1:500 dilution), LIN28B (Cell Signaling, #4196, 1:1000 dilution), LIN28A (Cell Signaling, #3978, 1:1000 dilution), SMAD2/3 (Cell Signaling, #8685, 1:1000 dilution), β-actin (Abcam, ab8227, 1:200,000 dilution), GAPDH (Santa Cruz, sc-137179, 1:10,000 dilution). Following incubation with the specific HRP-conjugated antibody (Dako, #P0447 or #P0448, 1:2,500 dilution), chemiluminescence signal was detected using Amersham^TM ECL^TM Western blotting detection reagents (GE Healthcare) and Amersham Hyperfilm^TM ECL (GE Healthcare). Uncropped scans of the most important blots are shown in Supplementary Fig. 13.

The siRNAs against SNAI1, MMP9 and INHBA (all from Santa Cruz Biotechnology, USA) were used at 20 nM/well using 2 μl Lipofectamine RNAiMAX (Invitrogen) in the cells seeded in 6-well plates. The RNA interference against PITX2 was carried out by the ON-TARGET plus SMART pool siRNA at 20 nM/well using 2 μl of Dharmafect-1 transfection reagent (Dharmacon) in cells seeded in 6-well culture plates. After 48 h of transfection, the cells were harvested for RNA/protein isolation. When required, rhTGF-β1 was added after 24 h of respective siRNA transfection into the cells.