8.0 μm transparent pet membrane

Py2T-LT cells (TGFβ > 20 days) were transfected for 2 days with siRNA against Bcl9 and/or Bcl9L or control siRNA (siCtr). In total, 2.5 10⁴ cells were plated onto a 0.2–20% FBS/DMEM Gradient medium in a 24-well Boyden chamber plate with 8.0 μm Transparent PET Membrane (Corning, NY, USA) and incubated for 18 h at 37 °C. Cells on the membrane were fixed with 4% paraformaldehyde and counterstained with DAPI (1 μg/ml; Sigma D9542). Non-migrated cells from the upper surface of the transwell membrane were removed by scrapping with a cotton swab. Migrated cells were imaged from the bottom of the Transwell membrane with a 10x objective on a Leica DMI microscope and quantified with ImageJ software.

The transwell assay was performed to assess cell invasion ability. HTR‐8 cells were seeded in 24‐well plates with Falcon® Permeable Support for 24‐well plates and an 8.0 μm Transparent PET Membrane (Corning, USA). HTR‐8 cells were added in 200 μL FBS‐free medium and placed in the upper chamber of each insert covered with Matrigel (Corning). The lower chamber was filled with 500 μL medium containing 10% FBS. The plate was incubated at 37°C for 48 h. Cells were then fixed in 4% paraformaldehyde at room temperature followed by permeabilization with 100% methanol for 5 min and crystal violet staining for 20 min. Images were analysed and quantified by Image J.