Figure S2.
TA cells control R-spondin production in the crypt niche. (A) qRT-PCR of Dll4, Tgf-β, and Egf in the whole intestinal tissue from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d after tamoxifen treatment. (B) qRT-PCR of Wnt3 and Wnt3a mRNA levels in whole intestinal tissue from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment (n = 3). (C) Representative pictures of IF of Wnt3a in intestinal samples from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d after tamoxifen treatment. (D) WB from of samples from whole intestine from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d after tamoxifen treatment. Membranes are blotted with indicated antibodies. (E) WB quantification from D. (F) WB from HCT-116 cells following URI knockdown by silencing RNA. Additionally, cells were treated with increasing concentrations of human R-spondin 1. Membranes are blotted with indicated antibodies. (G) WB quantification from F. (H) Representative pictures of in situ hybridization (ISH) for R-spondin 1 in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 3). Embryonic mouse kidney was used as positive control. (I) Quantification for H in number of R-spondin 1 positive cells per bottom part of the crypt (n = 3). (J) Representative pictures of IHC for lysozyme and ISH for R-spondin 1 in intestinal sections from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment (n = 3). Orange arrows point to positive cells. (K) Representative pictures of IF for lysozyme 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 lysozyme-positive cells per crypt in K after 2, 4, and 6 d of tamoxifen treatment (n = 3). (M) WB analysis of different concentrations of purified recombinant human R-spondin 1 and whole intestinal lysates from C57BL/6 mice (s.e., short exposure; l.e., long exposure) (N) Representative pictures of IF of R-spondin 1 in HCT-116 (irradiated with 40 Gy) and SW620 (nonirradiated) colorectal cell lines treated or not with siRNA against human R-spondin 1. (O) WB from HCT-116 (irradiated with 40 Gy) and SW620 (nonirradiated) cells following R-spondin 1 knockdown by using silencing RNA. Membranes are blotted with indicated antibodies. (P) IF of R-spondin 1 in R-spondin 1(+/+) and R-spondin 1(−/−) mouse embryo. (Q) IF of R-spondin 1 of whole mounted organoids transfected with siRNA control (siCtrl) or siRNA against human R-spondin 1 (siRspo1), 48 h after transfection. (R) Representative pictures of IF for R-spondin 1 and Gomori’s trichrome staining in carbamylcholine and aceclidine-treated mice. (S) Representative pictures of IF for endogenous R-spondin 1 and Gomori’s trichrome staining in IL-13–treated mice. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Scale bars represent 5 µm in J; 10 µm in H; 20 µm in C, K, N, R, and S; and 50 µm in P and Q. WB are representative of at least three independent experiments and IF and IHC are representative of at least three independent mice. Source data are available for this figure: SourceData FS2.

TA cells control R-spondin production in the crypt niche. (A) qRT-PCR of Dll4, Tgf-β, and Egf in the whole intestinal tissue from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d after tamoxifen treatment. (B) qRT-PCR of Wnt3 and Wnt3a mRNA levels in whole intestinal tissue from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment (n = 3). (C) Representative pictures of IF of Wnt3a in intestinal samples from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d after tamoxifen treatment. (D) WB from of samples from whole intestine from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d after tamoxifen treatment. Membranes are blotted with indicated antibodies. (E) WB quantification from D. (F) WB from HCT-116 cells following URI knockdown by silencing RNA. Additionally, cells were treated with increasing concentrations of human R-spondin 1. Membranes are blotted with indicated antibodies. (G) WB quantification from F. (H) Representative pictures of in situ hybridization (ISH) for R-spondin 1 in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment (n = 3). Embryonic mouse kidney was used as positive control. (I) Quantification for H in number of R-spondin 1 positive cells per bottom part of the crypt (n = 3). (J) Representative pictures of IHC for lysozyme and ISH for R-spondin 1 in intestinal sections from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment (n = 3). Orange arrows point to positive cells. (K) Representative pictures of IF for lysozyme 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 lysozyme-positive cells per crypt in K after 2, 4, and 6 d of tamoxifen treatment (n = 3). (M) WB analysis of different concentrations of purified recombinant human R-spondin 1 and whole intestinal lysates from C57BL/6 mice (s.e., short exposure; l.e., long exposure) (N) Representative pictures of IF of R-spondin 1 in HCT-116 (irradiated with 40 Gy) and SW620 (nonirradiated) colorectal cell lines treated or not with siRNA against human R-spondin 1. (O) WB from HCT-116 (irradiated with 40 Gy) and SW620 (nonirradiated) cells following R-spondin 1 knockdown by using silencing RNA. Membranes are blotted with indicated antibodies. (P) IF of R-spondin 1 in R-spondin 1(+/+) and R-spondin 1(−/−) mouse embryo. (Q) IF of R-spondin 1 of whole mounted organoids transfected with siRNA control (siCtrl) or siRNA against human R-spondin 1 (siRspo1), 48 h after transfection. (R) Representative pictures of IF for R-spondin 1 and Gomori’s trichrome staining in carbamylcholine and aceclidine-treated mice. (S) Representative pictures of IF for endogenous R-spondin 1 and Gomori’s trichrome staining in IL-13–treated mice. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Scale bars represent 5 µm in J; 10 µm in H; 20 µm in C, K, N, R, and S; and 50 µm in P and Q. WB are representative of at least three independent experiments and IF and IHC are representative of at least three independent mice. Source data are available for this figure: SourceData FS2.

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