S7023

We isolated organoids, used tamoxifen to induce mosaic E-cad deletion, and then washed two times using PBS without Ca²⁺ or Mg²⁺. The organoids were trypsinized to small cell clusters by incubating with TrypLE for 10–15 minutes at 37° C. Cells were resuspended in PBS (-Ca2+, -Mg2+), filtered through a 40 μm filter, cell number estimated, and resuspended again at a density of 2×10⁶ cells/mL. The cell populations were then analyzed on a Beckman-Coulter MoFlo Cytometer. Cells were sorted for the expression of mT/ mG. Additional gating was performed to sort populations with higher forward and side scatter, corresponding to 2–4 cell clusters. The nozzle size for sorting clusters was 100 μm. Dead cells were excluded in the analysis by gating cells that stained negative for propidium iodide. Cells were sorted into organoid media and were resuspended in Matrigel and cultured in 24-well plates with FGF2-containing organoid media, supplemented with 50 nM of tamoxifen, for 7 days. The number of colonies formed in each well was assessed using a bright-field microscope. Small molecule and protein reagents used in this study included: an apoptosis inhibitor (z-VAD-FMK, SelleckChem S7023), a TGFbR1 inhibitor (SB525334; SelleckChem S1476), N-acetyl cysteine (Sigma, A7250), and soluble TGFβ1 (PeptoTech 100–21).