Fig 2: Loss of MIST1 expression does not affect epithelial proliferation or migration. (A) Top panel: IdU was given 90 minutes before death to monitor proliferation by labeling cells in S phase. Scale bar equals 100 μm. Bottom panel: CldU was given 24 hours before death to evaluate migration of cells. Scale bar equals 100 μm. (B) Quantification of percentage of IdU+ cells per crypt. n = 3 mice per group. (C) Quantification of average cell position of highest CldU+ cells along the crypt/villus axis. n = 3 mice per group.

Fig 3: Histological analysis of SMG-ECM-treated cultures reveals that aggregates display an expression pattern different from monolayer cells. A Paraffin sections of cells from 14 day cultures (i.e., untreated BM-MSCs [Untreated], SMG-ECM-treated aggregates [Agg/SMG-ECM], and monolayer [Mono/SMG-ECM] cells) were prepared and stained with H&E or PAS. PAS-positive cells are identified with arrows in the figure. Bar: 50 µm. B Immunofluorescence staining of cells prepared as in A and rat SMG tissue (positive control) was used to evaluate the presence and localization of amylase, Aqp5, Mist1, Krt14, Cldn3, and Cldn10 protein. Staining with nonspecific isotype antibody was used as a negative control (not shown). Bar: 50 µm

Fig 4: Jagged1 and Mist1 are localized at the base of gastric corpus, and Jagged1 stimulates Mist1 expression in stomach cancer cells. (A) Anti-Jagged1 immunostaining in the gastric corpus of WT and KrasLSL-G12D/+;Pdx1-cre (KC) mice at 1 month of age. Arrows: base of gastric corpus. (B) Double immunofluorescence staining for Jagged1 and Mist1 in the gastric corpus of WT mice at 3 weeks of age. (C) Quantitative RT-PCR for HES1, HES5, HEY1, HEY2, and MIST1 in Hs746T cells cultured in plates precoated with control Fc or Jagged1-Fc protein. (D) Quantitative RT-PCR for HES1, HES5, HEY1, HEY2, and MIST1 in SNU1 cells cultured in plates precoated with control Fc or Jagged1-Fc protein. Scale bars: 50 μm. *P ≪ .05, **P ≪ .005.

Fig 5: Intermediate cell phenotype of Mist1–/–mice is not due to impaired Notch signaling. (A) Mist1+/+ and Mist1–/– mice were treated with DBZ to inhibit Notch signaling, and jejunal tissues were stained with alcian blue. Scale bar equals 125 μm. (B) Jejunal tissues from (A) were stained with PTAB to evaluate Paneth cells (yellow arrows) and intermediate cells (black arrows). Paneth cell granules from Mist1+/+ Paneth cells stain brown, and granules from Mist1–/– Paneth cells that contain mucins stain purple, suggesting an intermediate cell phenotype. Scale bar equals 50 μm. (C) Quantification of Paneth cell and intermediate cell numbers. n = 4 mice per group. ****P <.0001. **P <.01.