Fig 1: Eph receptor inhibition does not alter auditory vs. vestibular targeting.(A) RDA tracing combined with neurofilament immunofluorescence was used to determine whether auditory axons were correctly targeted. An example of EphB1-Fc treated embryo with normal targeting is shown. (B) Example of EphA3-Fc treated embryo with normal auditory targeting. (C) Similarly, treatment with EphA4-Fc did not alter central targeting of auditory VIIIth nerve axons. (D) Targeting of vestibular axons was determined in embryos that showed GFP transfection in vestibular, but not auditory, ganglion cells. Axons were followed through brainstem sections. Transfection in this example was with EphA4; all GFP labeled axons were found in vestibular projection regions. (E) Transfection with EphB2 plasmid did not alter central vestibular VIIIth nerve projections. (F) Misexpression of kiEphA4 did not alter central vestibular projections. (G) Similarly, kiEphB2-transfected embryos showed no mistargeting of vestibular axons into auditory projection regions. Scale bar, 100 µm in A-C and 300 µm in D-G.
Fig 2: Eph receptor inhibition causes reduction in hindbrain compartment.(A-D) Neurofilament immunofluorescence (left) of hindbrain sections at level of VIIIth nerve entry with auditory (black) and vestibular (gray) components schematized (right). Red dashed line indicates entire dorsoventral extent of the VIIIth nerve projections in hindbrain. Volumes found in IgG-Fc (A) EphB1-Fc (B), and EphA3-Fc (C) did not significantly differ. (D) Vestibular hindbrain volume is reduced following EphA4-Fc treatment. (E,F) Graphs of mean auditory (E) and vestibular (F) volumes for all treatment groups. Numbers of samples are indicated in each bar; asterisk indicates p <0.05.
Fig 3: WCDD301 enhances intracellular F-actin and EphA4 intensities in both human and mouse dispersed islet cells.(A) Immunostained images of human dispersed islet cells for F-actin and glucagon in vehicle-treated (C, control) and cells treated with WCDD301 or Ephrin-A5 Fc (E). (B) Immunostained images of mouse dispersed islet cells for F-actin and glucagon in C, 301, and E groups. (C) Immunostained images of human dispersed islet cells for EphA4 and glucagon in C, 301, and E groups. (D) Immunostained images of mouse dispersed islet cells for EphA4 and glucagon in C, 301, and E groups. (E) Comparison of F-actin intensity among C, 301, and E groups of human cells. (F) Comparison of F-actin intensity among C, 301, and E groups of mouse cells. (G) Comparison of EphA4 intensity among C, 301, and E groups of human cells. (H) Comparison of EphA4 intensity among C, 301, and E groups of mouse cells. On the scatter bar plot, each dot represents values (mean ± SD) of 1 mouse or human. Values (mean ± SEM) of treatment groups (n = 3 human, and n = 5 mouse) were compared with the respective controls using 1-way ANOVA, α = 0.05. *P < 0.05, **P < 0.01. Scale bar: 10 μm.
Fig 4: High binding affinity of WCDD301 for EphA4 receptor in a competitive way that suppresses glucagon secretion without side effect on insulin secretion.(A) Structural formula of WCDD301. (B) Determining binding affinity of WCDD301 for EphA4 without global fitting; each curve demonstrates best fit values of 3 individual replicates. (C) Determining binding affinity of WCDD301 for EphA4 via Cheng-Prusoff equation; each curve demonstrates best fit values of 3 individual replicates. (D) Demonstrating competition between WCDD301 and Ephrin-A5 Fc concentrations for binding to a fixed level of EphA4 (3 μg/mL) in ELISA system via AUC analysis. Values (mean ± SD) of AUC at 2 nM (0.025 ± 0.0011 AU), 4 nM (0.0469 ± 0.0014 AU), 6 nM (0.0554 ± 0.0006 AU), and 8 nM (0.0617 ± 0.0011 AU) of WCDD301 were compared; *** indicates difference of P < 0.001 between each successive concentration of Ephrin-A5 Fc (n = 3). (E) Competition between WCDD301 and Ephrin-A5 Fc for binding to a fixed level of EphA4 (3 μg/mL) through Michaelis-Menten kinetics; Ki values (n = 3) of WCDD301 challenges were compared with the 0 μM of WCDD301. Relative secretion of (F) glucagon and (G) insulin in murine dispersed islet cells in the presence and absence of WCDD301 or Ephrin-A5 Fc (E). Values of treatment groups were compared with the respective control; each dot demonstrates values (mean ± SD) of a single mouse (n = 5). (H) Secretion of glucagon in murine dispersed islet cells following cotreatment of WCDD301 with an EphA4 antagonist, rhyncophylline (Rhy). Values of treatment groups were compared with the corresponding controls; each dot demonstrates values (mean ± SD) of a single mouse (n = 5). In all experiments, values (mean ± SEM) were compared using 1-way ANOVA, α = 0.05. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5: WCDD301 recovers mononuclear cell infiltrate and restores F-actin and EphA4 intensities in islets of NOD diabetic mice.Hematoxylin-Eosin staining of pancreatic islets in (A) diabetic, (B) nondiabetic, and (C) WCDD301-treated mice. Immunostaining of islets for glucagon, F-actin, and EphA4 in (D) diabetic, (E) nondiabetic, and (F) WCDD301-treated mice (scale bar: 20 μm). Quantitation of (G) F-actin and (H) EphA4 signals in α cells of nondiabetic, diabetic, and WCDD301-treated mice. Values (mean ± SEM; n = 3) compared among groups using 1-way ANOVA. *P < 0.05, ***P < 0.001.
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