Fig. 6.

In crypt organoids, suppression of NF-κB activity results in strongly reduced growth, but also an increase in goblet cells and a loss of Paneth cells. (A) Quantification of viable organoid formation at days 4 and 8 after isolation of PSI crypts from control or ΔN mice (n=4 per group). ****P<0.0001 (unpaired t-test). Error bars represent s.e.m. (B) Quantification of organoid growth in the presence (WENR) or absence (ENR) of Wnt3 in culture medium at days 5 and 8 after isolation of PSI crypts from control or ΔN mice (n=3 per group). *P<0.05 (unpaired t-test). n.s., not significant. Error bars represent s.d. (C) Upper panels: Representative images of crypt organoids cultured in ENR medium (without Wnt) at day 1 (d1) and day 4 (d4) after isolation of PSI crypts from control and ΔN mice (n=4 per group). Black arrows indicate viable organoids. Lower panels: Representative images of crypt organoids cultured in WENR medium (plus Wnt) at day 1 (d1) and day 5 (d5) after isolation of PSI crypts from control and ΔN mice (n=3 per group). (D) Representative immunofluorescence staining using an anti-lysozyme antibody (green) and DAPI (blue) of control and ΔN crypt organoids at d2 or d7 in ENR medium. (E) Representative images of immunofluorescence staining using E-cadherin (E-Cad, red) and Muc2 (green) antibodies on control and ΔN crypt organoids at d2 in ENR or WENR medium, or of Alcian Blue staining of Lgr5-EGFP (control) and Lgr5-EGFP;ΔN (ΔN) organoids, generated from single Lgr5⁺ cells isolated by FACS, at d7 grown in WENR medium. Scale bars: 50 µm. (F,G) qRT-PCR for the Paneth cell marker lysozyme and the goblet cell marker Muc2 using RNA from organoids at d7 in ENR medium (F) or WENR medium (G). *P<0.05, ****P<0.0001 (multiple t-test with Bonferroni correction). Error bars represent s.e.m.