FIG 8.

Effects of the YAP inhibitor verteporfin on the phenotypes of Csk-deficient intestinal organoids. (A) Intestinal organoids from the jejunum of 8-week-old Csk CKO mice were cultured in the absence or presence of verteporfin (Verte) (1 μM) for 2 days and then subjected to immunostaining with antibodies to Ki67 (red) and to staining of nuclei with DAPI (blue) (left panel). Dashed lines indicate the boundary of intestinal organoids. Scale bar, 100 μm. The Ki67-positive area was also determined as a percentage of the total organoid area in such images (right panel). (B) Intestinal organoids cultured as for panel A for 3 days were subjected to immunostaining with antibodies to mucin 2 (red) and to E-cadherin (green) (left panel). Scale bar, 50 μm. The number of mucin 2-positive cells per bud was also determined in such images (right panel). (C) Intestinal organoids cultured as for panel A were examined by light microscopy at 3 days after plating (left panel). Boxed regions in the left images are shown at higher magnification in the right images. Arrowheads indicate granule-containing Paneth cells. Scale bars, 50 μm. The number of granule-containing Paneth cells per bud was also determined in such images (right panel). (D) Intestinal organoids cultured as for panel A were examined by light microscopy at 5 days after plating (left panel). Scale bar, 100 μm. The organoid area (middle panel) and the number of buds per organoid (right panel) were also determined in such images. Quantitative data are means ± SEM for a total of 75 organoids (A and D [middle panel]), 45 buds (B), 60 buds (C), or 60 organoids (D [right panel]) per group in three separate experiments. ***, P < 0.001 (Student's t test).