Fig. 5

Differential regulation of GSK3 in colon primitive cells from male and female mice. a Immunolabeling of Pser21/9 GSK3 in colonoids from male and female mice was performed at day 6 of culture. Fluorescence intensity of the brightest cross section of colonoids was quantified by Image J and is represented (n = 2 independent experiments). Representative images of immunolabeling of PserGSK3 are shown. Right panel: Western blot of Pser21/9 GSK3 and total GSK3β in colonoids is shown. Vinculin was used as loading control. Results are representative of two independent experiments. b Colonoids from male and female mice were incubated daily with GSK3 inhibitor (SB216763, 12.5 μM) from day 2 to day 6 of culture. Ki-67 labeling in male and female colonoids treated by GSK3 inhibitor was performed at day 6 of culture. The percentage of Ki-67 positive nuclei was calculated as described in the “Methods” section by the ratio of positive Ki-67 nuclei vs total nuclei in the larger diameter of colonoids. Representative colonoid labeling of Ki-67 (green) and nuclei by DAPI (cyan) is shown. Data are from two independent experiments