Supplementary Information Legends 803
Supplemental Figure 1 Differences in morphology between normal and 804 adenomatous mouse colon. 805
3D confocal imaging of tissues from normal mouse colon and Apcmin/+ mouse colon 806 adenoma showing DAPI in blue, Phalloidin (F-actin) in magenta, E-cadherin in green. 807 Confocal images of a section of the (a) normal epithelium and the (c) adenoma 808 epithelium indicates the differences in crypt morphology. (b) shows the 3D 809 reconstruction of (a) while panel d is a zoomed image of the boxed region in c. Colon 810 tissue was cleared and stained as described in ref 54. Briefly, the tissues were fixed, 811 cleared and stained with rat monoclonal anti-E-cadherin (Clone ECCD-2, Thermo 812 Fisher Scientific Cat #13-1900 (1:250)), goat anti-Rat IgG (H+L) Cross-Absorbed 813 Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific Cat #A-11006 814 (1:500)), Rhodamine Phalloidin (Molecular Probes/Invitrogen (1:200)) and DAPI 815 (Thermo Fisher Scientific Cat# 62248 (1:1000)). Images were acquired on a Leica SP8 816 Resonant Scanning Confocal microscope with the data processed/rendered using 817 Imaris software package (Bitplane, Zürich, Switzerland). Scale bar: (b) 100μm (c) 818 50μm. 819
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Supplemental Figure 2 Crypt formation occurs in another colon adenoma 821 organoid line 822
Colon adenoma organoids derived from other Apcmin/+ mice were tested for its crypt 823 formation capability using EGF and other ErbB ligands. Adenoma line #B13 organoids 824 were cultured, cultured with EGF or other ErbB ligands for up to 21 days. 825 Representative EDF images of crypt forming organoids from these #B13 cultures 826 stimulated with (a) 5 ng/mL EGF and (b) 0.5 μg/mL Neuregulin/Heregulin-β1. Inset 827 presents zoomed images of selected regions of the respective well on the left. Scale 828 bars: left panel 500 μm, right panels/inset 100 μm. 829
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Supplemental Figure 3 Workflow for colon adenoma organoid imaging 831 analyses 832
The workflow had been optimised to maximise the use of a 384well optical plate for 833 analysing the growth and morphology of the organoids. Colon adenoma fragments 834 were mixed with Matrigel 50% (v/v). This mixture was then equally divided into two 835 columns of a precooled 96well PCR plate sitting on a cold thermoblock. An automatic 836 liquid handling machine (Eppendorf epMotion® P5073c) was then used to transfer 8 837 μL per well, 8 wells at a time with an 8-channel pipetting tool. The plate was then 838 centrifuged to move all the cultures to the bottom of the wells before incubating at 839 room temperature for 1 hour. Similarly, media was prepared with the different 840 concentrations of the stock solutions of ligands and inhibitors were prepared in a 96 841 deep well plate and transferred (80 μL) to the adenoma cultures using the epMotion® 842 liquid handling machine. 843
ErbB signaling required for colon crypt formation Tan et al
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Supplementary Table 1 844
Summary of the IC50 values for the ErbB receptor kinase inhibitors.
