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Additional file 1: Fig. S1 General behavioral characteristics of GD mice. (A) Body weight (g), (B) body temperature (°C), (C) grip strength (Newton, N) (D) and wire hang latency (s) are represented. The p-values indicate genotype effect in one-way ANOVA. Values are means ± SEM. Suppl. Fig. S2. Barnes maze in GD mice. (A) Latency, (B) Number of errors, and (C) Distance traveled to first reach the target hole during the training session. The p-values indicate genotype effect in two-way repeated measures ANOVA. Values are means ± SEM. The test was conducted on a white circular surface, 1.0 m in diameter, with 12 holes equally spaced around the perimeter (O’Hara & Co., Tokyo, Japan). The circular open field was elevated 75 cm from the floor. A black Plexiglas escape box (17 × 13 × 7 cm), which had paper cage bedding at the bottom, was located under one of the holes. The hole above the escape box represented the target. The maze was rotated daily, with the spatial location of the target unchanged with respect to the distal visual room cues. Three trials per day were conducted for 5 days. One day and 8 days after the acquisition test, probe trials were conducted without the escape box. The number of errors, latency and distance traveled to reach the target hole and the time spent around each hole were recorded by Image BM software. Fig. S3. Probe test of Barnes maze in GD mice. (A) Time spent around the target hole in the probe test 1 day after the last training session (right panel). Latency, number of errors, and distance traveled to first reach the target hole are represented in left panel. (B) Time spent around the target hole in the probe test 8 days after the last training session (right panel). Latency, number of errors, and distance traveled to first reach the target hole are represented in left panel. The p-values indicate genotype effect in two-way repeated measures ANOVA (A; left panel, B; left panel) or one-way ANOVA (A; three panels on the right, B; three panels on the right). Values are means ± SEM. Fig. S4. Brain weight and macroscopic architecture in GD mice. There is no obvious difference in the total weight (A) and macroscopic architecture (B) of the brain between WT and GD mouse (n = 3, p = 0.4871, t-test). (C) Golgi staining of spines in CA1 region. There is no obvious difference in CA1 region between WT and GD mouse brains. Fig. S5. Detection of trans-dimer in WT or GD mutant N-cadherin. (A) Scheme of the experiment methods to detect N-cadherin trans-dimer. Either NcadWT(GD)-HA or NcadWT(GD)-Flag were separately transfected into HEK293 cells. 24 after transfection, cells were scraped off. Then HEK293 cells transfected with NcadWT(GD)-HA and with NcadWT(GD)-Flag were co-cultured for another 48hrs to allow formation of trans-dimer (dimer formed by cell–cell adhesion). Cells were harvested and N-cadherin trans-dimer were detected by immunoprecipitation with anti-HA antibody followed by western blot. (B) Cell lysates were pulled down with anti-HA anti-body, followed by the blotting with anti-Flag antibody. (C) N-cadherin trans-dimer quantified by normalization with total N-cadherin and β-actin, mean + SD (p = 0.8274 n = 4, t-test). Fig. S6. Intact basal synaptic transmission in hippocampal CA1 region of GD mice. Schaffer collateral/commissural fiber-CA1 pyramidal cell synaptic transmission was examined in the presence of the NMDAR antagonist D-APV. GD mice showed no changes in the dependence of fiber volley amplitude (left) and EPSP slope (right) on stimulus intensity. Table S1. |
