Figure S3.
R-spondin supplementation restores Lgr5highISC proliferation. (A–C) qRT-PCR of Axin 2 and c-Myc (A), Ascl2 and Olfm4 (B), and Mki67 and Aurkb (C) in sorted Lgr5low cells from untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3, 3). (D) Representative pictures of IHC of γH2AX, p53, p21, cleaved caspase 3, and caspase 1 in intestinal sections 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. (E) qRT-PCR of Atoh1, Hes1, and Gfi1 in sorted- Lgr5low cells from untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3, 3). (F) Representative pictures of IF of lysozyme, IHC of chromogranin A, alkaline phosphatase staining, and Alcian Blue/PAS staining in intestinal sections from untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice; and corresponding quantifications (n = 3). (G) Experimental design for in vivo tracing of URI expressing cells in URI-CreERT2-EGFP; LSL-Katushka mice at indicated time points. (H) Representative picture of co-IF of Sox9 and Katushka and quantification of Sox9−/Katushka+ cells in URI-CreERT2-EGFP;LSL-Katushka mice. (I) Experimental design for in vivo tracing of URI expressing cells in URI(+/lox)Int; URI-CreERT2-EGFP; LSL-Katushka mice at indicated time points. (J) Representative picture of co-IF of Sox9 and Katushka and quantification of Sox9−/Katushka+ cells in URI(+/lox)Int; URI_CreERT2-EGFP; LSL_Katushka mice after 6 d of tamoxifen treatment. (K) Representative images of stemness assay of sorted Lgr5high and Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP after 7 d. (L) Quantification of sphere number in percentage from cultured Lgr5high and Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP after 7 d from K. (M) Quantification of sphere diameter (µM) from Lgr5high cells from J (n = 3). 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 D, H, and J; 75 μm in K; and 100 μm in F. IF and IHC are representative of at least three independent mice.

R-spondin supplementation restores Lgr5 high ISC proliferation. (A–C) qRT-PCR of Axin 2 and c-Myc (A), Ascl2 and Olfm4 (B), and Mki67 and Aurkb (C) in sorted Lgr5low cells from untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice, and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3, 3). (D) Representative pictures of IHC of γH2AX, p53, p21, cleaved caspase 3, and caspase 1 in intestinal sections 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. (E) qRT-PCR of Atoh1, Hes1, and Gfi1 in sorted- Lgr5low cells from untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3, 3). (F) Representative pictures of IF of lysozyme, IHC of chromogranin A, alkaline phosphatase staining, and Alcian Blue/PAS staining in intestinal sections from untreated URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice and R-spondin 1–treated URI(Δ/Δ)Int-Lgr5-EGFP mice; and corresponding quantifications (n = 3). (G) Experimental design for in vivo tracing of URI expressing cells in URI-CreERT2-EGFP; LSL-Katushka mice at indicated time points. (H) Representative picture of co-IF of Sox9 and Katushka and quantification of Sox9−/Katushka+ cells in URI-CreERT2-EGFP;LSL-Katushka mice. (I) Experimental design for in vivo tracing of URI expressing cells in URI(+/lox)Int; URI-CreERT2-EGFP; LSL-Katushka mice at indicated time points. (J) Representative picture of co-IF of Sox9 and Katushka and quantification of Sox9−/Katushka+ cells in URI(+/lox)Int; URI_CreERT2-EGFP; LSL_Katushka mice after 6 d of tamoxifen treatment. (K) Representative images of stemness assay of sorted Lgr5high and Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP after 7 d. (L) Quantification of sphere number in percentage from cultured Lgr5high and Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP after 7 d from K. (M) Quantification of sphere diameter (µM) from Lgr5high cells from J (n = 3). 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 D, H, and J; 75 μm in K; and 100 μm in F. IF and IHC are representative of at least three independent mice.

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