Fig. 4.

Post-screen validation of KDAC inhibitors AK-7, oxamflatin, and CI-994 potentiating effect on Arc protein abundance. a Western blot analysis reveals that lysates from BDNF-treated cortical cultures supplemented with AK-7, oxamflatin, or CI-994 have significantly more total Arc protein than BDNF plus vehicle (DMSO) control condition. Chemical structure of each compound is presented on top. b Graphs show mean (n = 5) Arc/β-actin ratio (±SEM) for cells treated as in a. One-way ANOVA revealed a significant dose–response difference between compound concentrations (AK-7, F _2,12 = 4.48, p < 0.05; oxamflatin, F _2,12 = 46.72, p < 0.0001; CI-994, F _2,12 = 12.24, p < 0.005). Tukey’s HSD post hoc test, *p < 0.05; **p < 0.01, ***p < 0.005; ****p < 0.0001. Western blots run with the same sample lysates revealed that these three KDAC inhibitors do not alter BDNF-dependent phosphorylation of p44/42 Mapk and rpS6. c Each tested KDAC inhibitor increases number of Arc puncta along dendrites labeled by Map2 immunocytochemistry. Representative captures of DIV14 mouse primary cortical neurons from each experimental condition co-immunostained for Arc (red fluorophore) and Map2 (green fluorophore). White arrowheads show examples of discrete Arc puncta. Primary cortical neurons were treated with BDNF and AK-7, oxamflatin, or CI-994 at a final concentration of 16.7 µM. Scale bar 10 µm. d Graph shows mean Arc puncta per dendrite µm for cells treated as in c. Separate ANOVA revealed a significant difference between cells co-treated with a KDAC and BDNF over untreated and BDNF alone conditions (AK-7, F _2,82 = 12.82, p < 0.0001; oxamflatin, F _2,78 = 46.31, p < 0.0001; CI-994, F _2,82 = 8.08, p < 0.001). Tukey’s HSD post hoc test, *p < 0.05; ***p < 0.005; ****p < 0.0001