Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: Design and characterization of cell type specific Wnt signaling enhancing mimetic molecules. ( A ) Scheme of designed molecules. Fragments of RSPO2 spanning the Fu1 and Fu2 domains (action module) were fused the C-terminus of cell type specific or control scFv (targeting module). Specific mutations used are indicated. ( B ) In vitro STF activity of four RSPO proteins. Furin domains of human RSPO1-4 were tested as fusions to an anti-GFP scFv. RSPO2 furin domain alone (N37-E143) was used as a control. Luciferase activity unit is arbitrary (RLU). ( C ) STF activity of RSPO mimetics on HEK293 ( top ) or HuH-7 ( bottom ) in the presence ( left ) or absence ( right ) of an exogenous Wnt source (30% Wnt3a conditioned media). ( D ) Quantitative PCR analysis of ASGR1 , ASGR2 and TFRC gene expression in HEK293, HuH-7 and A431 cells. The signals were relative to ACTB . ( E ) Western Blot analysis on LRP6 receptor and DVL2 phosphorylation in HuH-7 cells. Asterisk indicates a non-specific band that is detected in HuH-7 by the antibody used. The lower bands indicated by the arrow correspond to DVL2 with and without phosphorylation. Tubulin (TUB) is used as the loading control. 10% Wnt3a conditioned media was used as the exogenous Wnt source. Images were cropped for clarity. The uncropped images are presented in Fig. .

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: In Vitro, Activity Assay, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: Confirmation of receptor-specific activation of Wnt signaling. ( A ) STF activity in HEK293 cells transiently transfected with TFR1 (control, top ), ASGR1 ( middle ), and ASGR1 together with ASGR 2 cDNAs ( bottom ), either in the presence ( left ) or absence ( right ) of exogenous Wnt source (30% Wnt3a conditioned media). ( B ) STF activity in A431 cells with TFR1 or ASGR1 overexpression as specified. ( C ) Flow cytometry analysis of cell surface level of FZD proteins. HEK293 cells were transiently transfected with either ZNRF3 alone ( top ) or ASGR1 and ZNRF3 cDNAs ( bottom ), treated with RSPO mimetics as specified at 10 nM, then stained by the pan-FZD antibody 18R5.

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: Activation Assay, Activity Assay, Transfection, Over Expression, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: In vivo tissue-specific enhancement of Wnt signaling. ( A ) STF activity of the RSPO mimetic molecules in the appended-IgG format. ( B ) BLI analysis of the ASGR1 antibody (expressed as a Fab) on human and mouse ASGR1. The affinity (Kd) determined by steady-state fitting is indicated. ( C ) Axin2 and ( D ) Ki67 expression in liver ( upper panel ) and small intestine ( lower panel ) were analyzed by quantitative PCR, 48 h after i.p. injection of proteins as specified (n = 8 mice per group). Statistical analysis was performed using 1-way ANOVA: (ns) not significant, ** p < 0.01, **** p < 0.0001. ( E ) Immunofluorescence staining of liver samples for Ki67 and HNF4α from 10 mg/kg treatment groups. White arrows denote some double-positive cells as examples. DAPI was used to stain nuclei.

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: In Vivo, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Injection, Immunofluorescence, Staining