Figure 2.

aISCs undergo apoptosis and expulsion from the crypt base after exposure to DXR. (A) Representative images of CASP3 immunofluorescence co-localizing with Lgr5^eGFP+ cells in a jejunal crypt at 6 hours and 24 hours after DXR as compared with control. Scale bar = 10 μm. (B) Quantification of the percentage of CASP3⁺ cells that are Lgr5^eGFP+ or non-Lgr5^eGFP+ within the crypt epithelium at 6 hours and 24 hours after DXR as a percentage of total CASP3⁺ cells/crypt (mean ± SD; n = 3 per time point, 10 crypts/mouse). ∗∗P < .01; 2-way analysis of variance followed by Bonferroni’s post hoc test. (C) Combined GFP fluorescence and differential inference contrast time lapse of 1 representative Lgr5^eGFP+ enteroid after DXR. Scale bar = 50 μm. Within the inset image of the Lgr5^eGFP+ bud, the asterisk indicates the autofluorescent lumen and the dotted line identifies Lgr5^eGFP+ cells present initially in the enteroid bud, which are subsequently extruded into the lumen over time after DXR application. The yellow double-headed arrow is an example of the measurements performed in (D). Two-dimensional deconvolution was applied to the GFP channel. (D) Quantification of measured distance (μm) of maximal GFP⁺ intensity (representing Lgr5^eGFP+ cells) from the basolateral membrane of the enteroid bud over time after DXR or vehicle (media) application to Lgr5^eGFP+ enteroids. 4 ng/μL DXR (n = 7 GFP⁺ enteroid buds) or vehicle (n = 4 GFP⁺ enteroid buds) was applied in vitro and enteroids were imaged by time-lapse microscopy. Data are presented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; repeated-measures 2-way analysis of variance followed by Holm-Sidak post hoc test.