Figure 7.

ATF2 and ATF7 are required to protect epithelial cells against TNF-induced death in organoid culture. Colonic epithelium was isolated from Atf2^wt/wtAtf7^+/+ and Atf2^-/-Atf7^ko/ko mice and cultured as organoids (n = 3, independent organoid cultures). (A) qRT-PCR for the floxed region of Atf2 and Atf7 in colonic organoids. (B) Representative brightfield images of colonic organoids 72 hours after reseeding. (C) Quantification of organoid area and perimeter. (D) EdU incorporation assay using flow cytometry 72 hours after reseeding single cells (n = 3 for 2 independent experiments). (E) qRT-PCR for stem cell markers Lgr5 and Olfm4. (F) PI viability assay on colon organoids challenged with TNF-α for 12 hours. Scale bar: 250 μm. Red fluorescence indicates cell death. (G) Percentage of nonviable organoids quantified per well (n = 2). Graph bars show means and SEM, ∗∗P < .01, ∗∗∗P < .001, Student t test.