Figure 6.
URI deletion-mediated cell death decreases R-spondin production and tissue regeneration. (A) Scheme summarizing the mechanisms and processes occurring in TA cells in URI-depleted mice and possible R-spondin level regulation. (B) Representative pictures of co-IF for GFP and cleaved caspase 3 in non-irradiated and irradiated URI(+/+)Int-Lgr5-EGFP. (C) qRT-PCR of Uri, Rspo1, and Rspo3 mRNA levels in samples from intestines of non-irradiated URI(+/+)Int-Lgr5-EGFP and URI(+/Δ)Int-Lgr5-EGFP, and irradiated URI(+/+)Int-Lgr5-EGFP, URI(+/Δ)Int-Lgr5-EGFP mice (n = 3, 3, 3, 4). (D) Experimental design for chronic irradiation experiments in URI(+/+)Int and URI(+/Δ)Int mice. (E) Mice body weight depicted in percentage related to initial body weight from non-irradiated URI(+/+)Int and URI(+/Δ)Int mice and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice (n = 5 per group). (F) Intestinal permeability checked by FITC/Dextran assay in non-irradiated URI(+/+)Int and URI(+/Δ)Int mice and in 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice, after 6 wk of treatment (n = 5). (G) Representative pictures of H&E staining, IHC of URI, γH2AX, p53 and cleaved caspase 3 and Sirius red staining. (H) URI protein level quantification from non-irradiated URI(+/+)Int and URI(+/Δ)Int mice, and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). (I) Quantification of γH2AX positive cells from non-irradiated URI(+/+)Int and URI(+/Δ)Int mice, and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). (J) Quantification of p53 positive cells from nonirradiated URI(+/+)Int and URI(+/Δ)Int mice, and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). (K) Villi length in micrometers from nonirradiated URI(+/+)Int and URI(+/Δ)Int mice and 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA. Scale bars represent 20 µm in B; 50 µm in G; and 100 µm in G (H&E panels). IF and IHC are representative of at least three independent mice.

URI deletion-mediated cell death decreases R-spondin production and tissue regeneration. (A) Scheme summarizing the mechanisms and processes occurring in TA cells in URI-depleted mice and possible R-spondin level regulation. (B) Representative pictures of co-IF for GFP and cleaved caspase 3 in non-irradiated and irradiated URI(+/+)Int-Lgr5-EGFP. (C) qRT-PCR of Uri, Rspo1, and Rspo3 mRNA levels in samples from intestines of non-irradiated URI(+/+)Int-Lgr5-EGFP and URI(+/Δ)Int-Lgr5-EGFP, and irradiated URI(+/+)Int-Lgr5-EGFP, URI(+/Δ)Int-Lgr5-EGFP mice (n = 3, 3, 3, 4). (D) Experimental design for chronic irradiation experiments in URI(+/+)Int and URI(+/Δ)Int mice. (E) Mice body weight depicted in percentage related to initial body weight from non-irradiated URI(+/+)Int and URI(+/Δ)Int mice and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice (n = 5 per group). (F) Intestinal permeability checked by FITC/Dextran assay in non-irradiated URI(+/+)Int and URI(+/Δ)Int mice and in 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice, after 6 wk of treatment (n = 5). (G) Representative pictures of H&E staining, IHC of URI, γH2AX, p53 and cleaved caspase 3 and Sirius red staining. (H) URI protein level quantification from non-irradiated URI(+/+)Int and URI(+/Δ)Int mice, and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). (I) Quantification of γH2AX positive cells from non-irradiated URI(+/+)Int and URI(+/Δ)Int mice, and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). (J) Quantification of p53 positive cells from nonirradiated URI(+/+)Int and URI(+/Δ)Int mice, and from 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). (K) Villi length in micrometers from nonirradiated URI(+/+)Int and URI(+/Δ)Int mice and 5 Gy chronically irradiated URI(+/+)Int and URI(+/Δ)Int mice after 6 wk of treatment (n = 5). Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA. Scale bars represent 20 µm in B; 50 µm in G; and 100 µm in G (H&E panels). IF and IHC are representative of at least three independent mice.

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