Phenotypic alterations in TA cells modulate Lgr5 high ISC proliferation. (A) Scheme describing the generation of URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice by crossing URI lox mouse with Villin-creERT2 and Lgr5-EGFP-IRES-creERT2 mice. (B) Experimental design for BrdU labeling in vivo in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice at indicated time points. (C–E) Co-IF for EGFP and BrdU in intestinal sections from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment, representative pictures (C), quantification of total BrdU positive cells (D) and quantification of total Lgr5+, Lgr5−/BrdU+, and Lgr5+/BrdU+ cells per crypt(E). (F) qRT-PCR of Mki67 and Aurkb mRNA levels in intestinal Lgr5high sorted cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ) Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. Results are expressed as fold change (n = 10, 7). (G) qRT-PCR of Ddit4, Nfe2l2, Kdm5b, Jarid2, Cdkn1b, and Mex3a mRNA levels in sorted Lgr5high cells from URI(+/+)Int-Lgr5-GFP and URI(Δ/Δ)Int-Lgr5-GFP mice (n = 6, 6). (H) Representative IF for Mex3a in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (I) Mex3a positive cell number per crypt from H. (J) Representative plot from flow cytometry for quiescent cells (Pyronin Y) in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (K) Quantification of quiescent cells (Lgr5high and Pyronin Ylow) in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (L) Quantification of intensity in FSC-A channel from gated-Lgr5high cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 3). Data represent mean ± SEM; L depicts geometric mean (GeoMean) ± 95% confident interval (CI); *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Scale bars are 20 µm in C; and 100 µm in H. IF is representative of at least three independent mice.
