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Functional modifications in ZNRF3 induced by species with different testicular positions and amino acid changes at residue 406. a) to c) Presents the total biding energy (ΔG) of molecular docking between ZNRF3- and DVL1, DVL2, and DVL3, with lower ΔG values indicating stronger binding affinity. d) S406G mutation enhances the inhibitory effect of ZNRF3 on Wnt signaling pathway. Relative luciferase activity was measured using the TOPFlash reporter assay. The EV group represents cells transfected with an empty vector without <t>Wnt3a</t> treatment, while the Control group refers to empty vector-transfected cells treated with Wnt3a (60 ng/mL). All other groups were treated with Wnt3a. Data are displayed as mean ± SD ( n > 3). (**** P < 0.0001; *** P < 0.001; ns, not significant). e) Representative images of scratch assays performed on Hela cells expressing ZNRF3 wild-type and mutant variants from human (S406G), dolphin (G406S), and elephant (G406S) at 0 and 48 h. All groups, except the empty vector (EV) group, were treated with 100 ng/mL Wnt3a. Dashed lines indicate the wound edges, and cell migration was quantified by measuring wound closure at 48 h. Scale bar = 100 μm. f) Quantification of cell migration percentage (mean ± SD) across groups (one-way ANOVA, n = 3; * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant).
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Functional modifications in ZNRF3 induced by species with different testicular positions and amino acid changes at residue 406. a) to c) Presents the total biding energy (ΔG) of molecular docking between ZNRF3- and DVL1, DVL2, and DVL3, with lower ΔG values indicating stronger binding affinity. d) S406G mutation enhances the inhibitory effect of ZNRF3 on Wnt signaling pathway. Relative luciferase activity was measured using the TOPFlash reporter assay. The EV group represents cells transfected with an empty vector without Wnt3a treatment, while the Control group refers to empty vector-transfected cells treated with Wnt3a (60 ng/mL). All other groups were treated with Wnt3a. Data are displayed as mean ± SD ( n > 3). (**** P < 0.0001; *** P < 0.001; ns, not significant). e) Representative images of scratch assays performed on Hela cells expressing ZNRF3 wild-type and mutant variants from human (S406G), dolphin (G406S), and elephant (G406S) at 0 and 48 h. All groups, except the empty vector (EV) group, were treated with 100 ng/mL Wnt3a. Dashed lines indicate the wound edges, and cell migration was quantified by measuring wound closure at 48 h. Scale bar = 100 μm. f) Quantification of cell migration percentage (mean ± SD) across groups (one-way ANOVA, n = 3; * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant).

Journal: Molecular Biology and Evolution

Article Title: Evolutionary Dampening of Wnt Signaling May Contribute to Naturally Ascrotal Testes in Mammals

doi: 10.1093/molbev/msaf310

Figure Lengend Snippet: Functional modifications in ZNRF3 induced by species with different testicular positions and amino acid changes at residue 406. a) to c) Presents the total biding energy (ΔG) of molecular docking between ZNRF3- and DVL1, DVL2, and DVL3, with lower ΔG values indicating stronger binding affinity. d) S406G mutation enhances the inhibitory effect of ZNRF3 on Wnt signaling pathway. Relative luciferase activity was measured using the TOPFlash reporter assay. The EV group represents cells transfected with an empty vector without Wnt3a treatment, while the Control group refers to empty vector-transfected cells treated with Wnt3a (60 ng/mL). All other groups were treated with Wnt3a. Data are displayed as mean ± SD ( n > 3). (**** P < 0.0001; *** P < 0.001; ns, not significant). e) Representative images of scratch assays performed on Hela cells expressing ZNRF3 wild-type and mutant variants from human (S406G), dolphin (G406S), and elephant (G406S) at 0 and 48 h. All groups, except the empty vector (EV) group, were treated with 100 ng/mL Wnt3a. Dashed lines indicate the wound edges, and cell migration was quantified by measuring wound closure at 48 h. Scale bar = 100 μm. f) Quantification of cell migration percentage (mean ± SD) across groups (one-way ANOVA, n = 3; * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant).

Article Snippet: After 24 to 48 h of transfection, the cells were treated with Wnt3a Surrogate Protein (MedChemExpress) or control treatments.

Techniques: Functional Assay, Residue, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Reporter Assay, Transfection, Plasmid Preparation, Control, Expressing, Migration