Figure 5. Brd4 Suppression by RNAi and JQ1 Inhibits Intestine Organoid Formation.

(A) Representative images of intestine crypt cultures from CAG^rtTA3-expressing TtG-Ren.713 and TtG-Brd4.552 mice. Animals were fed a dox diet for 2 weeks and intestine crypts maintained on dox in vitro (left), crypts cultured off dox in vitro (center), or animals were fed dox-diet for 2 weeks, withdrawn for 2 weeks, and maintained off dox in vitro (right).

(B) Quantification of organoid-forming efficiency for cultures shown in (A).

(C) Representative images of C57Bl/6 intestine crypts when cultured in the presence of DMSO or 100 nM JQ1.

(D) Quantification of organoid-forming efficiency for crypt cultures shown in (C).

(E) Western blot of Brd4 protein levels in intestinal villi from CAG^rtTA3-expressing TtG-Ren.713, TtG-Brd4.552, and TtG-Brd4.1448 mice, maintained on a dox diet for 2 weeks (+) or treated with dox for 2 weeks and withdrawn for 2 weeks (+/−). The expression of GFP and β-Actin are also shown.

(F) H&E, BrdU, Keratin 20 (KRT20), lysozyme (Paneth cell), OLFM4 (stem cell), and Alcian blue (goblet cell) staining of small intestine in CAG^rtTA3-expressing TtG-Ren.713 and TtG-Brd4.552 mice fed a dox diet for 2 weeks and in Brd4.552 mice following 2 weeks of dox withdrawal. Asterisks (*) indicate a statistically significant difference between shRen.713 and shBrd4.552 organoid-forming efficiency (p < 0.05), as determined by two-tailed Student’s t test.

(G) Quantification of Alcian blue-positive goblet cells in CAG^rtTA3-expressing TtG-Ren.713 and TtG-Brd4.552 mice after 2 weeks on dox and CAG^rtTA3/+; TtG-Brd4.552 mice taken off dox for 2 weeks. Error bars represent the mean of three independent samples ± SEM. Data in (B) and (D) represent mean ± SD (n ≥ 3).