Fig. 4 | Nature Communications

Fig. 4

From: Anti-LRP5/6 VHHs promote differentiation of Wnt-hypersensitive intestinal stem cells

Fig. 4

Characterization of anti-LRP5/6 VHHs with highest potency. a IC50 calculations of inhibition of cellular responses to Wnt3a by titration of the indicated anti-LRP6P3 VHHs in a luciferase reporter assay. Mean luciferase activities ± s.d. (n = 3) are plotted. b IC50 calculations by combination of Wnt3a and Rspo1. Mean luciferase activities ± s.d. (n = 2) are plotted. c Activation of Wnt signaling mediated by CHIR-99021 is not affected by anti-LRP6P3 VHHs. Wnt luciferase reporter assay performed in HEK293T cells treated with 5 µM CHIR-99021 and 10 μM of the indicated anti-LRP6P3 VHHs. Graph shows average (bars) and range (dots) of luciferase activity in duplicate cell cultures transfected in parallel. d Wnt luciferase reporter assay performed in LRP6-/- HEK293T cells. Wnt pathway activation achieved upon LRP5 overexpression is counteracted by anti-LRP5/6P3 VHHs. Graph shows average (bars) and range (dots) of luciferase activity in duplicate cell cultures transfected in parallel. e Western blot showing inhibition of Wnt3a-induced LRP6 phosphorylation (pLRP6) by the different anti-LRP5/6P3 VHHs. f anti-LRP5/6P3 VHHs do not inhibit Wnt1-mediated cellular responses. Wnt luciferase reporter assay in HEK293T cells overexpressing Wnt1 and treated with 10 μM of the indicated anti-LRP5/6P3 VHHs. Graph shows average (bars) and range (dots) of luciferase activity in duplicate cell cultures transfected in parallel. g Top panels: Schematic representation of the growth conditions, associated phenotypes and experimental set up. Bottom panels: anti-LRP5/6P3 VHHs induce death of Wnt3a-treated wild type (WT) intestinal organoids. Organoids were cultured in WENR and treated with 10 μM of the indicated anti-LRP5/6P3 VHHs for 4 d (Scale bar, 400 μm). Red asterisks indicate cell death; green arrows indicate organoids showing villi and crypts; blue arrows indicate cystic organoids

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