Figure S1.
Phenotypic alterations in TA cells modulate Lgr5highISC proliferation. (A) Representative pictures of co-IF staining for Vimentin and URI in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after 6 d of tamoxifen treatment. (B) Time course qRT-PCR of Gfi1 and Hes-1 mRNA levels in sorted Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after 0, 2, 4, and 6 d of tamoxifen treatment (n = 3). (C) qRT-PCR of apoptotic (Apaf, Noxa, Bax, and Puma) and pyroptosis (Il1β) markers in sorted Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (D) Representative pictures of co-IF for EGFP and cleaved caspase 3 in intestinal sections from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (E) Quantifications of Lgr5−/cleaved caspase 3+ cell number per crypt in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after tamoxifen treatment. (F) Representative scatter plot from flow cytometry experiments of Lgr5high ISCs in isolated crypts from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (G) Percentage of Lgr5high ISCs in intestines from URI(+/+)nt-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after 6 d on tamoxifen treatment (n = 4). (H) Quantification of Lgr5+ cell number per crypt in the different part of the intestinal tract (duodenum, jejunum, and ileum) of URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 3). (I) Quantification of GFP fluorescence intensity of Lgr5-GFPhigh cells in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 3). (J) Quantification of GFP fluorescence intensity of Lgr5-GFPlow cells (n = 4, 3). (K) Representative pictures of IF for EGFP in intestinal sections from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (L) Quantification of Lgr5-GFP fluorescence intensity in bottom crypts of the intestine from K (n = 3). (M) Quantification of Lgr5+/BrdU positive cells per crypt in the different part of the intestinal tract (duodenum, jejunum, and ileum) from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 3). (N) β-galactosidase (SA-β-Gal) staining in intestinal sections from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment. PanIN lesions were used as positive control. (O) qRT-PCR of Tnfrsf19 and Sox17 mRNA levels in Lgr5high sorted cells in intestines from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 5, 3, 5, 3). (P) Representative IHC of p19 ARF in intestinal sections from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment. Testis were used as positive control. (Q) Kaplan–Meier curve of URI(Δ/Δ)Int mice (red solid line; n = 4) and URI(Δ/Δ)Int; p16/p19(Δ/Δ) (dashed red line; n = 4) mice. (R) H&E staining in intestinal sections from URI(Δ/Δ)Int; p16/p19(Δ/Δ) mice at time of death. (S) Kaplan-Meier curve of URI(+/+)Int; p21(Δ/Δ) (black solid line; n = 5), URI(Δ/Δ)Int; p21(Δ/Δ) (green solid line; n = 8) and URI(Δ/Δ)Int; p21(+/+) (red solid line; n = 7) mice. (T) Representative pictures of H&E staining in intestinal sections from URI(+/+)Int; p21(Δ/Δ) and URI(Δ/Δ)Int; p21(Δ/Δ) mice at the time of death (8–12 d). Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; Student’s t test and Mantel–Cox. Scale bars represent 10 μm in D and P; 50 μm in K; and 100 μm in A, R, and T. IF, H&E, and IHC are representative of at least three independent mice.

Phenotypic alterations in TA cells modulate Lgr5 high ISC proliferation. (A) Representative pictures of co-IF staining for Vimentin and URI in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after 6 d of tamoxifen treatment. (B) Time course qRT-PCR of Gfi1 and Hes-1 mRNA levels in sorted Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after 0, 2, 4, and 6 d of tamoxifen treatment (n = 3). (C) qRT-PCR of apoptotic (Apaf, Noxa, Bax, and Puma) and pyroptosis (Il1β) markers in sorted Lgr5low cells from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (D) Representative pictures of co-IF for EGFP and cleaved caspase 3 in intestinal sections from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (E) Quantifications of Lgr5−/cleaved caspase 3+ cell number per crypt in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after tamoxifen treatment. (F) Representative scatter plot from flow cytometry experiments of Lgr5high ISCs in isolated crypts from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice. (G) Percentage of Lgr5high ISCs in intestines from URI(+/+)nt-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice after 6 d on tamoxifen treatment (n = 4). (H) Quantification of Lgr5+ cell number per crypt in the different part of the intestinal tract (duodenum, jejunum, and ileum) of URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 3). (I) Quantification of GFP fluorescence intensity of Lgr5-GFPhigh cells in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 3). (J) Quantification of GFP fluorescence intensity of Lgr5-GFPlow cells (n = 4, 3). (K) Representative pictures of IF for EGFP in intestinal sections from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (L) Quantification of Lgr5-GFP fluorescence intensity in bottom crypts of the intestine from K (n = 3). (M) Quantification of Lgr5+/BrdU positive cells per crypt in the different part of the intestinal tract (duodenum, jejunum, and ileum) from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 3). (N) β-galactosidase (SA-β-Gal) staining in intestinal sections from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment. PanIN lesions were used as positive control. (O) qRT-PCR of Tnfrsf19 and Sox17 mRNA levels in Lgr5high sorted cells in intestines from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 5, 3, 5, 3). (P) Representative IHC of p19 ARF in intestinal sections from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment. Testis were used as positive control. (Q) Kaplan–Meier curve of URI(Δ/Δ)Int mice (red solid line; n = 4) and URI(Δ/Δ)Int; p16/p19(Δ/Δ) (dashed red line; n = 4) mice. (R) H&E staining in intestinal sections from URI(Δ/Δ)Int; p16/p19(Δ/Δ) mice at time of death. (S) Kaplan-Meier curve of URI(+/+)Int; p21(Δ/Δ) (black solid line; n = 5), URI(Δ/Δ)Int; p21(Δ/Δ) (green solid line; n = 8) and URI(Δ/Δ)Int; p21(+/+) (red solid line; n = 7) mice. (T) Representative pictures of H&E staining in intestinal sections from URI(+/+)Int; p21(Δ/Δ) and URI(Δ/Δ)Int; p21(Δ/Δ) mice at the time of death (8–12 d). Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; Student’s t test and Mantel–Cox. Scale bars represent 10 μm in D and P; 50 μm in K; and 100 μm in A, R, and T. IF, H&E, and IHC are representative of at least three independent mice.

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