Figure 3.
Decreased R-spondin levels reduce ISC proliferative capacity. (A) qRT-PCR for Ki67 and Mex3a in sorted ISC Rspo, Dll4, and TGFβ in sorted Paneth cells and Rspo3 in whole intestinal tissue from URI(Δ/Δ)Int-Lgr5-EGFP mice. (B) Scheme of time course curves for Ki67, Mex3a, Rspo1, Dll4, TGFβ, and Rspo3 from A. (C) Schematic representation for Tet-regulated miR-E shRspo1 expression vector in a lentiviral backbone (REVIR-shRspo1). PGK promoter drives the constitute expression of mVenus-IRES-rtTA3 cassette, and Tet-responsive element promoter (T3G) induces the expression of dsRFP fluorescent protein-coupled miR-E shRspo1 or miR-E shRenilla upon doxycycline treatment. (D) Endogenous fluorescence showing the induction of dsRFP after doxycycline treatment (24 h). (E) Sorting strategy for mouse stable cell lines expressing REVIR-shRspo1 construct. (F) qRT-PCR showing Rspo1 levels in sorted cells following doxycycline treatment (48 h). (G) R-spondin 1 titration (0, 100, 200, 300, 400, and 500 nM) in mouse organoids. (H) Scheme of organoid transduction with REVIR-shRspo1 lentiviral particles and differentiation/co-culture experiments. (I) Morphology and GFP expression of organoids transduced with REVIR-shRspo1 lentiviral particles and culture in ENR-CV for 72 h. (J) Morphology of organoids derived from co-culture experiments between lentiviral transduced Paneth cells (with REVIR-shRenilla or REVIR-shRspo1) and untraduced ISCs. (K) qRT-PCR depicting stem cell markers (Egfp, RFP), Rspo1, and mKi67 in shRenilla and shRspo1-transduced organoids. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Scale bars represent 10 µm in D; 150 µm in I and J; and 200 µm in G. IF and bright-field photos are representative of at least three independent experiments.

Decreased R-spondin levels reduce ISC proliferative capacity. (A) qRT-PCR for Ki67 and Mex3a in sorted ISC Rspo, Dll4, and TGFβ in sorted Paneth cells and Rspo3 in whole intestinal tissue from URI(Δ/Δ)Int-Lgr5-EGFP mice. (B) Scheme of time course curves for Ki67, Mex3a, Rspo1, Dll4, TGFβ, and Rspo3 from A. (C) Schematic representation for Tet-regulated miR-E shRspo1 expression vector in a lentiviral backbone (REVIR-shRspo1). PGK promoter drives the constitute expression of mVenus-IRES-rtTA3 cassette, and Tet-responsive element promoter (T3G) induces the expression of dsRFP fluorescent protein-coupled miR-E shRspo1 or miR-E shRenilla upon doxycycline treatment. (D) Endogenous fluorescence showing the induction of dsRFP after doxycycline treatment (24 h). (E) Sorting strategy for mouse stable cell lines expressing REVIR-shRspo1 construct. (F) qRT-PCR showing Rspo1 levels in sorted cells following doxycycline treatment (48 h). (G) R-spondin 1 titration (0, 100, 200, 300, 400, and 500 nM) in mouse organoids. (H) Scheme of organoid transduction with REVIR-shRspo1 lentiviral particles and differentiation/co-culture experiments. (I) Morphology and GFP expression of organoids transduced with REVIR-shRspo1 lentiviral particles and culture in ENR-CV for 72 h. (J) Morphology of organoids derived from co-culture experiments between lentiviral transduced Paneth cells (with REVIR-shRenilla or REVIR-shRspo1) and untraduced ISCs. (K) qRT-PCR depicting stem cell markers (Egfp, RFP), Rspo1, and mKi67 in shRenilla and shRspo1-transduced organoids. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Scale bars represent 10 µm in D; 150 µm in I and J; and 200 µm in G. IF and bright-field photos are representative of at least three independent experiments.

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