Journal: Cellular and Molecular Neurobiology

Article Title: CREB2 Functions as a Central Mediator of Oxidative Neuronal Death Triggered by Microglial Glutamate Release Under Neuroinflammatory Conditions

doi: 10.1007/s10571-026-01695-w

Figure Lengend Snippet: p53 inhibition suppresses glutamate-induced CREB2 activation and downstream cytotoxic signaling. A HT22 cells were pretreated with 10 µM PFTα (a p53 inhibitor) for 1 h, followed by 5 mM glutamate (Glu) exposure for an additional 24 h. Protein levels of CREB2 and GADD45α were assessed by Western blotting and quantified (left panels). Cell viability was measured using the MTT assay (right panel). B Immunofluorescence staining of CREB2 was performed following the same treatment as in ( A ). Representative images show nuclei stained with Hoechst and CREB2 with FITC (scale bar = 50 μm). C Cells were transfected with p53 siRNA (sip53) for 24 h, followed by 5 mM glutamate treatment for an additional 24 h. Western blotting for CREB2 and p53 and their quantification (left panels) are shown. Cell viability was determined using the MTT assay (right panel). D Cells were transfected with CREB2 siRNA (siCREB2) for 24 h prior to glutamate treatment (5 mM, 24 h). Western blotting was performed to assess expression of phosphorylated p53 (Ser15), CREB2, and GADD45α (left panels). Quantification is shown in the left panel. Cell viability was assessed by MTT assay (right panel). E Cells were transfected with GADD45α siRNA (siGADD45α) for 24 h, followed by glutamate treatment (5 mM, 24 h). Protein levels of CREB2 and GADD45α were analyzed by Western blotting and quantified. Data are presented as mean ± SD ( n = 3). GAPDH served as a loading control. Statistical significance was determined using the following tests: One-way ANOVA with Bonferroni’s post hoc test for MTT in ( A ) [F(3, 8) = 333, p < 0.0001], ( C ) [F(3, 8) = 282, p < 0.0001], and ( D ) [F(3, 8) = 269.6, p < 0.0001]; and Two-way ANOVA with Bonferroni’s post hoc test for WB quantification in ( A ) [F(3, 16) = 437.4, p < 0.0001], ( C ) [F(3, 16) = 12.85, p = 0.0002], ( D ) [F(3, 24) = 83.83, p < 0.0001], and ( E ) [F(3, 16) = 1054, p < 0.0001]. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant

Article Snippet: After three washes, cells were incubated with FITC-conjugated goat anti-mouse secondary antibody (1:200; Jackson ImmunoResearch, cat. no. 115-095-003) for 1 h. Nuclei were counterstained with 5 μM Hoechst 33,342 (stock solution diluted immediately prior to use; Thermo Fisher Scientific, Cat. No. H1399) for 5 min at room temperature.

Techniques: Inhibition, Activation Assay, Western Blot, MTT Assay, Immunofluorescence, Staining, Transfection, Expressing, Control