Journal: Advanced Science

Article Title: Deficiency of Leucine‐Rich Repeat Containing G Protein‐Coupled Receptor 4 in Pancreas Reduces β Cell Mass

doi: 10.1002/advs.202508858

Figure Lengend Snippet: LGR4 regulates islet β cell proliferation via the Wnt‐β‐catenin‐ Ccnd1 axis. Data are presented as mean±SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 versus the negative control, #### p <0.0001 versus other groups. Data in (A, I, K) were analyzed by One‐way ANOVA, data in J were analyzed by Two‐way ANOVA, and data in the other panels were analyzed by t‐test. INS‐1 cells were used to explore the mechanism by which LGR4 regulates islet β cell proliferation, n = 3. A) Transfection efficiency of all three strains of rat Lgr4 siRNA. siRNA #3 was used for subsequent experiments; B–D) INS‐1 cells were transfected with Lgr4 siRNA ( Lgr4 KD) and control siRNA (Ctrl), and treated with glucose (GLU) or mannitol control (MNT) for 48 h, relative Ccnd1 mRNA levels (B), cell viability measured by CCK‐8 assay (C), and representative EdU staining (D); E, F) Nuclear β‐catenin levels of Lgr4 KD and Ctrl INS‐1 cells incubated for 48 h; G, H) Cytoplasm phosphorylated β‐catenin (Ser33/37/Thr41) levels of Lgr4 KD and Ctrl INS‐1 cells incubated for 48 h; I) Ccnd1 mRNA levels. INS‐1 cells were treated with different concentrations of human RSPO1 recombinant protein (RSPO1) for 24 h; J) Ccnd1 mRNA levels. Lgr4 KD and Ctrl INS‐1 cells were treated with or without 200 ng mL −1 RSPO1 for 24 h; K) Ccnd1 mRNA levels. INS‐1 cells were treated with different doses of IWR‐1 for 24 h. The mechanism was verified in transgenic mouse models. L, M) Nuclear β‐catenin levels of islets isolated from Lgr4 pko mice and Lgr4 fl/fl littermates fed on HFD, n = 3; N) Ccnd1 mRNA levels of islets isolated from Lgr4 pko mice and Lgr4 pko littermates fed on HFD, n = 6 and 5 for Lgr4 fl/fl and Lgr4 pko ; O–Q) Representative Ki‐67 and β‐catenin immunochemistry and quantification of iLgr4 bko mice and Lgr4 fl/fl littermates fed on HFD, n = 4.

Article Snippet: IWR‐1 (HY‐12238) and TRAF‐STOP inhibitor 6877002 (HY‐110247) were obtained from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Negative Control, Transfection, Control, CCK-8 Assay, Staining, Incubation, Recombinant, Transgenic Assay, Isolation

Journal: bioRxiv

Article Title: Proteomic analysis identifies the E3 ubiquitin ligase Pdzrn3 as a regulatory target of Wnt5a-Ror signaling

doi: 10.1101/2020.06.22.163790

Figure Lengend Snippet: A) Representative histogram showing the effect of rWnt5a treatment on NIH/3T3 GFP-Pdzrn3 (WRP) reporter cells. WRP cells were treated with rWnt5a (0.2μg/mL) for 6 hours, and GFP-Pdzrn3 fluorescence was measured by flow cytometry. B) Dose-response curve showing GFP-Pdzrn3 downregulation as a function of rWnt5a concentration in the WRP reporter assay. An EC 50 of 77.1ng/mL was calculated. C) Quantification of the effects of proteasome inhibitor (epoxomicin, 10μM), ubiquitin-activating enzyme E1 inhibitor (PYR41, 10μM) and Cullin inhibitor (MLN4924, 10μM) on rWnt5a-induced Pdzrn3 downregulation in the WRP reporter cells. Error bars represent ± SEM calculated from three technical replicates. t-test (unpaired) was performed to determine statistical significance for the following comparisons: inhibitors vs. the vehicle control DMSO. D) Quantification of the effects of canonical Wnt inhibitors, Dkk-1 (2μg/μL) and IWR-1-endo (10μM) on rWnt5a-induced Pdzrn3 degradation in the WRP reporter cells. Error bars represent ± SEM calculated from three technical replicates. t-test (unpaired) was performed to determine statistical significance for the following comparisons: inhibitors vs. the vehicle control DMSO. E) Quantification of the effects of Fzd1, Fzd2 and Fzd7 overexpression on the median fluorescence of WRP reporter cells. Error bars represent ± SEM calculated from two cell lines and three technical replicates per line. t-test (unpaired) was performed to determine statistical significance for the following comparisons: Fzd overexpression vs. the Myc epitope tag overexpression. F) Quantification of the effects of DVL1, DVL2 and DVL3 overexpression on the median fluorescence of WRP reporter cells. Error bars represent ± SEM calculated from two cell lines and three technical replicates per line. t-test (unpaired) was performed to determine statistical significance for the following comparisons: DVL overexpression vs. the Myc epitope tag overexpression. G) Quantification of the effects of CK1 inhibitor (D4476, 100μM) and GSK inhibitor (CHIR99021, 100μM) on rWnt5a-induced Pdzrn3 downregulation in the WRP reporter cells. Error bars represent ± SEM calculated from three technical replicates. t-test (unpaired) was performed to determine statistical significance for the following comparisons: inhibitors vs. the vehicle control DMSO. H) Quantification of the effect of genetically ablating Kif26a and Kif26b on rWnt5a-induced GFP-Pdzrn3 reporter degradation. Error bars represent ± SEM calculated from three technical replicates. t-test (unpaired) was performed to determine statistical significance for the following comparisons: GFP-Pdzrn3 reporter in Kif26a/Kif26b double KO cells vs. GFP-Pdzrn3 reporter in WT cells; GFP-Pdzrn3 reporter in Kif26b rescue cells vs. GFP-Pdzrn3 reporter in WT cells; GFP-Pdzrn3 reporter in Kif26a/Kif26b double KO cells vs. GFP-Pdzrn3 reporter in Kif26b rescue cells. I) Quantification of the effect of genetically ablating Pdzrn3 and Lnx4 on rWnt5a-induced GFP-Kif26b reporter (WRK) degradation. Error bars represent ± SEM calculated from three technical replicates per line. t-test (unpaired) was performed to determine statistical significance for the following comparisons: GFP-Kif26b reporter in Pdzrn3/Lnx4 double KO cells vs. GFP-Kif26b reporter in WT cells; GFP-Kif26b reporter in Pdzrn3 rescue cells vs. GFP-Kif26b reporter in WT cells; GFP-Kif26b reporter in Pdzrn3/Lnx4 double KO cells vs. GFP-Kif26b reporter in Pdzrn3 rescue cells. P-values: * = p<0.05, ** = p<0.01, *** = p<0.001.

Article Snippet: The following recombinant proteins and drugs were purchased: human/mouse Wnt5a (654-WN-010, R&D Systems); Wnt-C59 (C7641-2s; Cellagen Technology); epoxomicin (A2606, ApexBio); PYR-41 (B1492, ApexBio); MLN4924 (I50201M, R&D systems); mouse Dkk-1 (5897-DK-010, R&D Systems); IWR-1-endo (B2306, ApexBio); D4476 (A3342, ApexBio); and CHIR99021 (A3011, ApexBio).

Techniques: Fluorescence, Flow Cytometry, Concentration Assay, Reporter Assay, Over Expression

Journal: EvoDevo

Article Title: Wnt/β-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii

doi: 10.1186/s13227-018-0100-7

Figure Lengend Snippet: Wnt/β-catenin pathway is active in the larval gut and is affected by chemical manipulation. a Maximal projections and orthogonal virtual sections on the level of midgut and hindgut of a fluorescent confocal z -stacks of β-catenin protein immunostaining (green) on 7 dpf larvae done to confirm the chemical treatment’s efficacy. The larvae shown here come from the same batch as those used for in situ hybridization in Fig. . High levels of β-catenin were observed especially in the midgut. While the activation by CHIR99021 did not cause any dramatic increase in β-catenin levels, the inhibition of Wnt/β-catenin signalling by either of the inhibitors, JW55 or IWR-1-endo led to the complete absence of such high levels of β-catenin in the gut. b Fluorescent in situ hybridization of a putative Wnt target gene, Pdu - Axin (yellow) and quantification of the phenotype classes ( c ) shows no effect for the activator (CHIR99021) and mild effects of both Wnt/β-catenin inhibitors on axin expression. These larvae come from the same batch as those used for in situ hybridization in Fig. . d The fluorescent signal from the microinjected Wnt reporter construct SuperTOPFlash-tdTomato with 8 Tcf/LEF binding sites can be observed in the endoderm of 7 dpf transient transgenic larvae. This indicates that the Wnt/β-catenin signalling is active in the gut. e The effect of chemical manipulation of Wnt/β-catenin signalling on the expression of Pdu - Tcf

Article Snippet: We used CHIR99021 as an activator of the Wnt/β-catenin pathway (Biomedica) at 10 µM final concentration, since it works well on earlier (24–48 hpf) stages and higher concentrations often killed the larvae, and inhibitors JW55 (own stock, but available commercially) or IWR-1-endo (Merck Millipore 681669) at a 30 µM concentration.

Techniques: Immunostaining, In Situ Hybridization, Activation Assay, Inhibition, Expressing, Construct, Binding Assay, Transgenic Assay

Journal: EvoDevo

Article Title: Wnt/β-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii

doi: 10.1186/s13227-018-0100-7

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signalling converts the midgut to hindgut. a Fluorescent in situ hybridization of gut marker genes. b Quantification of the effect of activation/inhibition of Wnt/β-catenin pathway on the expression of these genes by assignment of all individuals to phenotypic classes. Pdu - Subtilisin - 1 (first row, red) , Pdu - Subtilisin - 2 (second row, magenta) , Pdu - α - Amylase (third row, cyan) = midgut marker genes, the darker the colour in the graph, the higher the expression. All three midgut marker genes show remarkably lower or no expression when Wnt/β-catenin signalling is inhibited (JW55 or IWR-1-endo). No pronounced effect was observed for activation of the pathway (CHIR99021). Pdu - Legumain (fourth row, yellow) = hindgut marker gene is normally highly expressed in the hindgut but only in very low levels in the midgut, which is also the case in the presence of activator, but it expands to midgut upon Wnt/β-catenin inhibition. Moreover, stronger inhibition (IWR-1-endo) causes it to be expressed outside the digestive tract in nephridia and other cells

Article Snippet: We used CHIR99021 as an activator of the Wnt/β-catenin pathway (Biomedica) at 10 µM final concentration, since it works well on earlier (24–48 hpf) stages and higher concentrations often killed the larvae, and inhibitors JW55 (own stock, but available commercially) or IWR-1-endo (Merck Millipore 681669) at a 30 µM concentration.

Techniques: Inhibition, In Situ Hybridization, Marker, Activation Assay, Expressing

Journal: EvoDevo

Article Title: Wnt/β-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii

doi: 10.1186/s13227-018-0100-7

Figure Lengend Snippet: Expression of endodermal marker genes. a Fluorescent in situ hybridization of two endodermal marker genes, Pdu - Nk2.1 and Pdu - Otx and b quantification of the effect of activation/inhibition of Wnt/β-catenin pathway on their expression by assignment of all individuals to phenotypic classes. The expression of these genes is typical for neuroectoderm in early larvae but similar to Pdu - Tcf , they obtain new expression domains in the gut later in development while still retaining their neurospecific expression. Pdu - Nk2.1 (top row, green) is expressed in both midgut and hindgut under normal circumstances, and the same pattern is observed in the presence of any of the tested inhibitors or the activator. This is consistent with the theory of midgut to hindgut conversion upon Wnt/β-catenin signalling inhibition since the gene is normally expressed in both tissues. Pdu - Otx is typical for mandibular cells, the entire midgut, whereas it is absent from most of the hindgut except for the very posterior cells which form the sides of the anus. Its midgut expression disappears in the presence of the stronger inhibitor (IWR-1-endo), while a normal pattern is observed with the milder JW55 inhibition (which might just point to unsuccessful inhibition on this batch). Neural and mandibular expression is preserved under all conditions, although it is somewhat lower after Wnt/β-catenin inhibition given the overall smaller size of mandibles. Interestingly, the cells surrounding the anus lose Pdu - Otx expression also in the presence of either CHIR99021 activator of Wnt/β-catenin signalling

Article Snippet: We used CHIR99021 as an activator of the Wnt/β-catenin pathway (Biomedica) at 10 µM final concentration, since it works well on earlier (24–48 hpf) stages and higher concentrations often killed the larvae, and inhibitors JW55 (own stock, but available commercially) or IWR-1-endo (Merck Millipore 681669) at a 30 µM concentration.

Techniques: Expressing, Marker, In Situ Hybridization, Activation Assay, Inhibition

Journal: EvoDevo

Article Title: Wnt/β-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii

doi: 10.1186/s13227-018-0100-7

Figure Lengend Snippet: Wnt/β-catenin signalling is necessary for cell proliferation and survival. a Proliferating cells labelled by incorporation of 5-ethynyl-2′-deoxyuridine (EdU, red) from 6 to 7 dpf and counterstained with DAPI (blue) to mark cell nuclei. Images are maximal projections of the whole fluorescent confocal z -stacks. Representative individuals are shown. b TUNEL staining of cell death (green), counterstained with Hoechst dye (blue) to mark nuclei. c Number of EdU positive proliferating cells in the whole larvae as counted manually on maximal projections of fluorescent z -stacks as those shown in ( a ). Averages are indicated in bold, n —number of individuals analysed per each experimental group, p — p value of a standard two tailed unpaired Student’s t test with unequal variance, s.d. —standard deviation. There are significantly less proliferating cells when IWR-1-endo is present. With the activator of Wnt/β-catenin signalling CHIR99021, there is no significant difference in the number of proliferating cells throughout the body. d Quantification of cell death performed in the same way as for proliferation, except only cells from the trunk up to midgut/pharynx boundary (or up to first pair of parapodia) were counted. There are significantly more dead or dying cells in groups treated with CHIR99021 or IWR-1-endo compared to control group which was treated with DMSO

Article Snippet: We used CHIR99021 as an activator of the Wnt/β-catenin pathway (Biomedica) at 10 µM final concentration, since it works well on earlier (24–48 hpf) stages and higher concentrations often killed the larvae, and inhibitors JW55 (own stock, but available commercially) or IWR-1-endo (Merck Millipore 681669) at a 30 µM concentration.

Techniques: TUNEL Assay, Staining, Two Tailed Test, Standard Deviation

Journal: EvoDevo

Article Title: Wnt/β-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii

doi: 10.1186/s13227-018-0100-7

Figure Lengend Snippet: Expression of Pdu - Cdx ParaHox gene. a 3D reconstructions of confocal fluorescent in situ hybridization’s z -stacks detecting a ParaHox gene Pdu - Cdx and b A graph showing the percentage of individuals assigned to classes according to the level of expression (the darker the higher) upon Wnt chemical treatment. Under control conditions, Pdu - Cdx is expressed in the whole hindgut, (in lower level) on the ventral floor of midgut and on the foregut/midgut border. With the Wnt activator (CHIR99021), it was expressed in similar levels but in entire gut, whereas with the inhibitor JW55, the expression is somewhat lower but the pattern remains the same. Widespread ectopic expression with the stronger inhibitor (IWR-1-endo) prevented the proper quantification of phenotypes when using bright-field microscopy. c Orthogonal views of the same z -stacks reveal the expression in the hindgut and at the midgut/foregut boundary connected by a narrow strip of Pdu - Cdx expression on the ventral floor of the gut. This expression expands to most of the gut upon Wnt activation, and the overall expression is lower in the presence of Wnt inhibitors

Article Snippet: We used CHIR99021 as an activator of the Wnt/β-catenin pathway (Biomedica) at 10 µM final concentration, since it works well on earlier (24–48 hpf) stages and higher concentrations often killed the larvae, and inhibitors JW55 (own stock, but available commercially) or IWR-1-endo (Merck Millipore 681669) at a 30 µM concentration.

Techniques: Expressing, In Situ, Microscopy, Stripping Membranes, Activation Assay