Figure 4.
R-spondin supplementation restores Lgr5highISC proliferation. (A) Representative organoids derived from URI(+/+)Int and URI(Δ/Δ)Int mice following 4 and 10 d in culture. Bright-field images are shown. The red arrow represents “crypt-like structures.” (B) Quantification of organoid growth in percentage at indicated time points in culture (n = 3; D, day). (C) Representative co-IF of URI and Sox9 in organoids from URI(+/+)Int and URI(Δ/Δ)Int mice following 10 d of culture. (D) qRT-PCR of Rspo1 mRNA levels in intestines from hURI(+/+)Int and hURI(+/KI)Int mice following 4 wk of doxycycline treatment (n = 6). (E) WB of isolated crypts from hURI(+/+)Int and hURI(+/KI)Int mice following 4 wk of doxycycline treatment. Membranes are blotted with the indicated antibodies. (F) Representative H&E staining and IHC of URI and Ki67 in organoids from hURI(+/+)Int and hURI(+/KI)Int mice under low (250 nM) R-spondin 1 concentration. (G) Representative co-IF of URI and R-spondin 1 in organoids from hURI(+/+)Int and hURI(+/KI)Int mice under low (250 nM) R-spondin 1 concentration. (H) Quantification of organoid growth from hURI(+/+)Int and hURI(+/KI)Int mice at indicated time points under low (250 nM) or normal (500 nM) R-spondin 1 concentration. Results are depicted as percentage of growth related to initial size (n = 3). (I) Experimental design for R-spondin 1 treatment in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (J) Representative co-IF for Lgr5-EGFP and BrdU in untreated URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (K) Quantification of Lgr5/BrdU double positive cells per crypt in percentage from J. (L) Representative scatter plot from flow cytometry experiments of quiescent cells in untreated URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (M) Quantification of quiescent cells (Lgr5high and Pyronin Ylow) in untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 5, 6, 5) from L. (N–P) qRT-PCR of Mki67 and Aurkb (N), Axin2 and c-Myc (O), and Ascl2 and Olfm4 (P) in sorted Lgr5high cells from URI(+/+)Int-Lgr5-EGFP, untreated URI(Δ/Δ)Int-Lgr5-EGFP, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3, 3). (Q) qRT-PCR of Mex3a mRNA levels in sorted Lgr5high cells from URI(+/+)Int-Lgr5-EGFP, untreated URI(Δ/Δ)Int-Lgr5-EGFP, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 4, 3). (R) Representative IF for Mex3a in intestinal tissue from URI(+/+)Int-Lgr5-EGFP, untreated URI(Δ/Δ)Int-Lgr5-EGFP, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice. (S) Mex3a-positive cell number per crypt from R. (T) Representative H&E staining of URI from untreated URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 8 d of tamoxifen treatment. (U) Kaplan–Meier curve of non-treated URI(Δ/Δ)Int-Lgr5-EGFP (red solid line; n > 3) and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP (black solid line; n > 3) mice. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test, one-way ANOVA and Mantel–Cox. Scale bars represent 20 µm in J; 50 µm in A, C, and G; and 100 µm in R and T. H&E, IF, and IHC are representative of at least three independent mice. Source data are available for this figure: SourceData F4.

R-spondin supplementation restores Lgr5 high ISC proliferation. (A) Representative organoids derived from URI(+/+)Int and URI(Δ/Δ)Int mice following 4 and 10 d in culture. Bright-field images are shown. The red arrow represents “crypt-like structures.” (B) Quantification of organoid growth in percentage at indicated time points in culture (n = 3; D, day). (C) Representative co-IF of URI and Sox9 in organoids from URI(+/+)Int and URI(Δ/Δ)Int mice following 10 d of culture. (D) qRT-PCR of Rspo1 mRNA levels in intestines from hURI(+/+)Int and hURI(+/KI)Int mice following 4 wk of doxycycline treatment (n = 6). (E) WB of isolated crypts from hURI(+/+)Int and hURI(+/KI)Int mice following 4 wk of doxycycline treatment. Membranes are blotted with the indicated antibodies. (F) Representative H&E staining and IHC of URI and Ki67 in organoids from hURI(+/+)Int and hURI(+/KI)Int mice under low (250 nM) R-spondin 1 concentration. (G) Representative co-IF of URI and R-spondin 1 in organoids from hURI(+/+)Int and hURI(+/KI)Int mice under low (250 nM) R-spondin 1 concentration. (H) Quantification of organoid growth from hURI(+/+)Int and hURI(+/KI)Int mice at indicated time points under low (250 nM) or normal (500 nM) R-spondin 1 concentration. Results are depicted as percentage of growth related to initial size (n = 3). (I) Experimental design for R-spondin 1 treatment in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (J) Representative co-IF for Lgr5-EGFP and BrdU in untreated URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (K) Quantification of Lgr5/BrdU double positive cells per crypt in percentage from J. (L) Representative scatter plot from flow cytometry experiments of quiescent cells in untreated URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (M) Quantification of quiescent cells (Lgr5high and Pyronin Ylow) in untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 5, 6, 5) from L. (N–P) qRT-PCR of Mki67 and Aurkb (N), Axin2 and c-Myc (O), and Ascl2 and Olfm4 (P) in sorted Lgr5high cells from URI(+/+)Int-Lgr5-EGFP, untreated URI(Δ/Δ)Int-Lgr5-EGFP, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3, 3). (Q) qRT-PCR of Mex3a mRNA levels in sorted Lgr5high cells from URI(+/+)Int-Lgr5-EGFP, untreated URI(Δ/Δ)Int-Lgr5-EGFP, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 4, 3). (R) Representative IF for Mex3a in intestinal tissue from URI(+/+)Int-Lgr5-EGFP, untreated URI(Δ/Δ)Int-Lgr5-EGFP, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice. (S) Mex3a-positive cell number per crypt from R. (T) Representative H&E staining of URI from untreated URI(Δ/Δ)Int-Lgr5-EGFP and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice following 8 d of tamoxifen treatment. (U) Kaplan–Meier curve of non-treated URI(Δ/Δ)Int-Lgr5-EGFP (red solid line; n > 3) and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP (black solid line; n > 3) mice. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test, one-way ANOVA and Mantel–Cox. Scale bars represent 20 µm in J; 50 µm in A, C, and G; and 100 µm in R and T. H&E, IF, and IHC are representative of at least three independent mice. Source data are available for this figure: SourceData F4.

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