Fig. 6: Effects of HPC on the level and distribution of USP22 in CA1 after tGCI.

A Immunohistochemistry of USP22 in the hippocampus after tGCI with or without hypoxia. Representative images show the sham-operated group (a, b), 26 h after reperfusion of tGCI groups (c, d) and HPC groups (e, f), 168 h after reperfusion of tGCI groups (g, h) and HPC groups (i, j), respectively. Scale bars: 250 μm (a, c, e, g, i) and 25 μm (b, d, f, h, j). B Quantitative analysis of USP22-positive cells in CA1. *p < 0.05 versus Sham animals (p = 0.345 in the hypoxia group, One-Way ANOVA Test; p < 0.001 in the tGCI group, One-Way ANOVA Test, p = 0.017 at tGCI 0 h, p < 0.001 at tGCI 4 h, p = 0.024 at tGCI 26 h, p = 0.001 at tGCI 50 h, p < 0.001 at tGCI 168 h, Tamhane post-test; p < 0.001 in the HPC group, One-Way ANOVA Test, p = 0.006 at HPC 26 h, p = 0.001 at HPC 50 h, p < 0.001 at HPC 168 h, Bonferroni post-test, n = 4-8), ^#p < 0.05 versus tGCI group at the same time point (p = 0.006 at 26 h, p = 0.023 at 50 h, p = 0.001 at 168 h, unpaired t-test). C Representative photomicrographs with fluorescent staining of USP22 (red), NeuN/GFAP/Iba-1 (green), and DAPI (blue) in CA1 after tGCI with or without hypoxia. Scale bars: 75 μm. D Western blot analysis of USP22 in CA1. GAPDH is used as loading control to normalize the relative expression of whole cellular USP22. Data are expressed as percentage of value of Sham animals. The histogram presents the quantitative analyses of USP22 levels. *p < 0.05 versus Sham animals (p = 0.536 in the hypoxia group, One-Way ANOVA Test; p = 0.019 in the tGCI group, One-Way ANOVA Test, p = 0.999 at tGCI 0 h, p = 0.982 at tGCI 4 h, p = 0.031 at tGCI 26 h, p = 0.045 at tGCI 50 h, Tamhane post-test; p < 0.015 in the HPC group, One-Way ANOVA Test, p = 0.136 at HPC 26 h, p = 0.014 at HPC 50 h, Bonferroni post-test, n = 3-5), ^#p < 0.05 versus tGCI group at the same time point (p = 0.103 at 26 h, p = 0.984 at 50 h, unpaired t-test). E, F Representative immunoblots and quantitative data show USP22 in cytoplasmic and nuclear fractions, separately. GAPDH and Lamin B1 are used as loading controls to normalize the relative expressions of cytoplasmic and nuclear USP22, respectively. Data are expressed as percentage of value of Sham animals (n = 4 in each group for cytoplasmic USP22, n = 6 for nuclear USP22). *p < 0.05 versus Sham animals (for cytoplasmic USP22, p = 0.016 at hypoxia 24 h, Kruskal-Wallis Test; p < 0.001 in the tGCI group, One-Way ANOVA Test, p = 0.502 at tGCI 0 h, p = 0.999 at tGCI 4 h, p = 0.018 at tGCI 26 h, p = 0.001 at tGCI 50 h, Tamhane post-test; p = 0.001 in the HPC group, One-Way ANOVA Test, p = 0.030 at HPC 26 h, p = 0.024 at HPC 50 h, Tamhane post-test, n = 4; for nuclear USP22, p = 0.121 in the hypoxia group, One-Way ANOVA Test; p < 0.001 in the tGCI group, One-Way ANOVA Test, p = 0.734 at tGCI 0 h, p = 1.000 at tGCI 4 h, p = 0.023 at tGCI 26 h, p = 0.024 at tGCI 50 h, Tamhane post-test; p = 0.466 in the HPC group, One-Way ANOVA Test, n = 6), ^#p < 0.05 versus tGCI group at the same time point (for cytoplasmic USP22, p = 0.894 at 26 h, p = 0.334 at 50 h, for nuclear USP22, p = 0.003 at 26 h, p = 0.025 at 50 h, unpaired t-test). G Immunoprecipitation blots showing the interaction between nuclear USP22 and nuclear β-catenin in the CA1 of tGCI and HPC groups. USP22 was immunoprecipitated (IP) using anti-USP22 antibody. IgG antibody was used as a negative control. β-catenin was detected by western blot. Data are expressed as percentage of Sham animals value. *p < 0.05 versus Sham animals (p = 0.005 in the tGCI group, p = 0.121 in the HPC group, n = 4, One-Way ANOVA Test, p = 1.000 at 26 h, p = 0.025 at 50 h, Bonferroni post-test), ^#p < 0.05 versus tGCI group at the same time point (p = 0.711 at 26 h, p = 0.006 at 50 h, unpaired t-test). Each bar represents the mean ± SD (error bars).