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. 2023 Aug 23;26(9):107707. doi: 10.1016/j.isci.2023.107707

Figure 4.

Figure 4

Enhanced protein SUMOylation in neuronal nuclei during seizures and Bic-induced AP bursting in cultured hippocampal neurons

(A) Representative images of DG, CA1, and CA3 pyramidal layer neurons in the hippocampus, and piriform cortex (PIR) of mock- and pilocarpine-injected animals immunolabelled with anti-SUMO-2/3 antibodies. SUN1-sfGFP (green); Hoechst (blue); SUMO-2/3 (red). Scale bars, 10 μm.

(B) Quantification of SUMO-2/3 nuclear intensity in neurons shown in (A). Nuclear intensities for SUMO-2/3 were measured from SUN1-sfGFP-positive nuclei outlined by Hoechst and SUN1-GFP staining. All values for pilocarpine-treatments are normalized against mean nuclear intensity from mock-injected animals. N = 3 pairs of animals, n = 75 number of cells quantified across all sections.

(C) Representative images of hippocampal neurons (DIV14 to 15) stimulated with Bic (40 μM) to induce AP bursting for 30 min and 24 h. SUMO-2/3 (red); Hoechst (blue); MAP2 (cyan). Scale bars, 5 μm.

(D) Quantification of SUMO-2/3 staining in hippocampal neurons under basal condition or stimulated conditions with Bic. All experimental values were normalized against mean nuclear intensity of SUMO-2/3 at basal condition for each independent experiment. N = 3 independent experiments, n = 60 total number of cells quantified across experiments.

(E) Representative Western blot of total SUMOylated proteins by SUMO-2/3. Total lane intensities for SUMO-2/3 were normalized against total protein amount measured with SYPRO Ruby protein blot stain. N = 4 pairs of animals. Paired t-test with means ± SEM, p < 0.05 (∗).

(F) Representative images of DG, CA1, and CA3 pyramidal neurons of the hippocampus, and piriform cortex (PIR) of mock- and pilocarpine-injected animals immunolabeled with anti-SUMO-1 antibodies. SUN1-GFP (green); Hoechst (blue); SUMO-1 (red). Scale bars, 10 μm.

(G) Quantification of SUMO-1 nuclear intensities as described in (B). N = 3 pairs of animals, n = 75 number of cells quantified across all sections.

(H) Representative images of hippocampal neurons (DIV14 to 15) stimulated with Bic (40 μM) to induce AP bursting for 30 min and 24 h. SUMO-1 (red); Hoechst (blue); MAP2 (cyan). Scale bars, 5 μm.

(I) Quantification of SUMO-1 nuclear intensity in hippocampal neurons under basal or Bic stimulation as described in (D). N = 3 independent experiments, n = 60 total number of cells quantified across experiments. Unless otherwise stated, all statistics for IHC and ICC experiments were performed using Mann-Whitney test with bar graphs reporting means ± SEM, p < 0.05 (∗), p < 0.01 (∗∗), p < 0.0001 (∗∗∗∗).