Figure 2.

SMAD4 expression in tumor organoids suppresses invasive capability. (A) Western Blot of doxycycline induced expression of SMAD4 in pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ tumor organoids. (B) Immunofluorescence images of wildtype organoids (Control) and untreated (−Dox) and treated (+Dox) pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ tumor organoids. (C) Representative images displaying growing cellular connections (white arrows) between tumor organoids in either untreated or treated conditions. Scale bar = 0.5 mm. (D) Smad4^KO BRAF^V600E/+ tumor organoids form significantly more connections than Smad4^KO BRAF^V600E/+ organoids with induced expression of Smad4 via doxycycline. (* = p-value = 0.0.017, Student’s t-test). 10× transmission and florescence images representative of 3 biological replicates. (E) EdU stain of tumor organoids treated with either vehicle or doxycycline. 10× images representative of 3 biological replicates (Scale bar = 0.5 mm). (F) Quantification of EdU-positive cells in pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ organoids.