Figure 12.

Effect of Ptpn2loss on mouse intestinal stem cell markers and enteroid growth. (A) Staining of intestinal stem cell marker Olfm4 in ileal tissue of whole-body Ptpn2-deficient mice (n = 3). (B) Western blot of ileal IECs probing for Olfm4. Per the antibody manufacturer, Olfm4 in the small intestine typically displays 2 bands that belong to Olfm4 protein, with molecular weight ranging from 85 to 90 and 70 kilodaltons. (C) Densitometry of both bands from Olfm4 protein level in ileal IECs. Columns show means ± SD. WT = 8, HET = 8, KO = 8. One-way analysis of variance and Tukey post hoc test. (D) RT-qPCR analysis of the Ascl2 gene, which is responsible for intestinal stem cell renewal in the crypts. WT = 9, HET = 11, KO = 10. Columns show geometric means ± geometric SD. One-way analysis of variance and Tukey post hoc test. (E) Enteroid cultures derived from small intestinal crypts of Ptpn2-WT and Ptpn2-KO mice showing enteroid size at days 1 and 4 after tissue harvest: WT = 2; KO = 4. (F) Counting of total number of viable enteroids on day 4. (G) Enteroid diameter on day 4. (H) Total number of budding enteroids on day 4 normalized by the number of total enteroids. Columns show means ± SD. Two-tailed unpaired t test. (I) Ptpn2 deletion was confirmed by TCPTP staining (green), 4′,6-diamidino-2-phenylindole (DAPI) (blue), and E-cadherin (red). (J) Bright-field microscopy of Ptpn2^fl/fl and Ptpn2^ΔIEC-derived enteroids induced with tamoxifen for 48 hours. (K) Number of budding enteroids before and 48 hours after tamoxifen induction. (L) Number of spheroids before and 48 hours after tamoxifen induction. TMX, tamoxifen.