Fig. 3: Effects of HPC on the levels of CUL1 and SCFKDM2A E3 ligase complex in CA1 after tGCI. | Communications Biology

Fig. 3: Effects of HPC on the levels of CUL1 and SCFKDM2A E3 ligase complex in CA1 after tGCI.

From: Inhibition of SCFKDM2A/USP22-dependent nuclear β-catenin ubiquitylation mediates cerebral ischemic tolerance

Fig. 3

A Immunohistochemistry of CUL1 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), respectively. Scale bars: 250 μm (a, c, e) and 25 μm (b, d, f). B Quantitative analysis of CUL1-positive cells in CA1. *p < 0.05 versus Sham animals (p = 0.391 in the Hypoxia groups, p < 0.001 in the tGCI groups, p = 1.000 at tGCI 0 h, p = 0.220 at tGCI 4 h, p = 0.014 at tGCI 26 h, p = 0.062 at tGCI 50 h, p < 0.001 at tGCI 168 h, One-Way ANOVA Test followed by Bonferroni post-test; p = 0.003 in the HPC groups, p = 1.000 at HPC 26 h, p = 1.000 at HPC 50 h, p = 0.035 at HPC 168 h, Kruskal Wallis Test, #p < 0.05 versus tGCI group at the same time point (p = 0.010 at 26 h, p = 0.145 at 50 h, p = 0.969 at 168 h, n = 4-6, unpaired t-test). C Western blot analysis of CUL1 in CA1. The histogram presents the quantitative analyses of CUL1 levels. GAPDH is used as loading control to normalize the relative expression of whole cellular CUL1. Data are expressed as percentage of value of Sham animals. *p < 0.05 versus Sham animals (p = 0.205 in the Hypoxia groups, p < 0.001 in the tGCI groups, p = 0.002 in the HPC groups, One-Way ANOVA Test, p = 0.732 at tGCI 0 h, p = 0.428 at tGCI 4 h, p = 0.044 at tGCI 26 h, p = 0.897 at tGCI 50 h, p = 0.434 for HPC 26 h, p = 0.011 for HPC 50 h, Tamhane post-test), #p < 0.05 versus tGCI group at the same time point (p = 0.001 at 26 h, p = 0.001 at 50 h, n = 4, unpaired t-test). D Representative photomicrographs with fluorescent staining of CUL1 (green), NeuN/GFAP/Iba-1 (red) and DAPI (blue) in CA1. Scale bars: 75 μm. E, F Representative immunoblots and quantitative data show SKP1 and CUL1 in cytoplasmic and nuclear fractions, respectively. GAPDH and Lamin B1 are used as loading controls to normalize the relative expressions of cytoplasmic and nuclear proteins of interest, respectively. Data are expressed as percentage of value of sham-operated animals. *p < 0.05 versus Sham animals (for cytoplasmic SKP1, n = 4, p = 0.056 for Hypoxia, p = 0.004 for tGCI, p = 0.443 for HPC, One-Way ANOVA Test, p = 1.000 at tGCI 0 h, p = 0.523 at tGCI 4 h, p = 0.042 at tGCI 26 h, p = 1.000 at tGCI 50 h; for cytoplasmic CUL1, n = 6, p = 0.616 for Hypoxia, p = 0.538 for tGCI, p = 0.376 for HPC, One-Way ANOVA Test; for nuclear SKP1, n = 4, p = 0.024 in the Hypoxia groups, p = 0.019 at Hypoxia 0 h, p = 0.517 at Hypoxia 24 h, Kruskal Wallis Test; p = 0.094 at tGCI 0 h, p = 0.249 at tGCI 4 h, p = 0.011 at tGCI 26 h, p = 0.070 at tGCI 50 h, unpaired t-test; for nuclear CUL1, n = 4, p = 0.005 in the Hypoxia groups, p = 0.048 at Hypoxia 0 h, p = 1.000 at Hypoxia 24 h, One-Way ANOVA Test followed by Tamhane post-test, p = 0.041 at tGCI 0 h, p = 0.355 at tGCI 4 h, p = 0.003 at tGCI 26 h, p < 0.001 at tGCI 50 h, unpaired t-test). #p < 0.05 versus tGCI group at the same time point (for cytoplasmic SKP1, p = 0.016 at 26 h, p = 0.152 at 50 h; for cytoplasmic CUL1, p = 0.933 at 26 h, p = 0.485 at 50 h; for nuclear SKP1, p = 0.016 at 26 h, p = 0.048 at 50 h; for nuclear CUL1, p = 0.006 at 26 h, p = 0.001 at 50 h; n = 4, unpaired t-test). G Immunoprecipitation assay showing the level of SCFKDM2A E3 ligase complex in CA1 of tGCI and HPC groups. KDM2A was immunoprecipitated (IP) using anti-KDM2A antibody. IgG antibody was used as a negative control. SKP1 and CUL1 was detected by western blot using anti-SKP1 and anti-CUL1 antibodies. Data are expressed as percentage of value of sham-operated animals. *p < 0.05 versus Sham animals (p = 0.034 for SKP1/KDM2A, p = 0.036 at tGCI 26 h, p = 0.201 at tGCI 50 h; p = 0.001 for CUL1/KDM2A, p = 0.001 at tGCI 26 h, p = 0.328 at tGCI 50 h, One-Way ANOVA Test followed by Bonferroni post-test), #p < 0.05 versus tGCI group at the same time point (n = 7 in each group for SKP1/KDM2A, p = 0.103 at 26 h, p = 0.041 at 50 h; n=6 in each group for CUL1/KDM2A, p = 0.009 at 26 h, p = 0.207 at 50 h, unpaired t-test). Each bar represents the mean ± SD (error bars).

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