Supplementary Figure 11: Characterization of AAV-Flag-dn-IκBα-p2A-eYFP viral vector, HDAC2 promoter assay in HEK293 cells, characterization of AAV-Cre viral vector and effect of chronic clozapine treatment on IκBα in the frontal cortex of Hdac2-cKO mice.
From: Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects
(a) Schematic representation of the recombinant AAV vector used to over-express both Flag-tagged dominant negative IκBα (IκBα-S32A-S36A; Flag-dn-IκBα) and eYFP under the CaMKIIα promoter in mouse frontal cortex pyramidal neurons. The arrowhead indicates p2A cleavage site. (b) Representative immunohistochemical images of mouse frontal cortex injected with the AAV-CaMKIIα::Flag-dn-IκBα-p2A-eYFP construct. Note that signals of Flag-dn-IκBα (red) and eYFP (green) do not overlap, indicating high cleavage efficiency of the p2A site. (c) AAV-CaMKIIα::Flag-dn-IκBα-p2A-eYFP was injected into the frontal cortex, and Flag expression was measured by Western blotting three weeks after surgery (Flag-dn-IκBα: ∼ 40 kDa). (d,e) Viral-mediated over-expression of eYFP in frontal cortex CaMKIIα-positive (b), but not PV-positive (e), neurons. (f) Neuro2A cells, which endogenously express CaMKIIα, were transfected with either AAV-CaMKIIα::eYFP (AAV-eYFP) or AAV-CaMKIIα::Flag-dn-IκBα-p2A-eYFP (AAV-Flag-dn-IκBα-p2A-eYFP). Western blot with anti-Flag antibody showed the expected molecular weight of Flag-dn-IκBα in cells transfected with AAV-Flag-dn-IκBα-p2A-eYFP, indicating high cleavage efficacy of the p2A peptide. (g) Neuro2A cells were transfected with the AAV-CaMKIIα::Flag-dn-IκBα-2A-eYFP construct. Red signal (Flag-dn-IκBα) that does not overlap with green signal (eYFP) indicates high cleavage efficacy of the p2A peptide. (h) Luciferase activity of HEK293 cells co-transfected with Flag-p65, or mock, together with either the HDAC2 promoter—luciferase construct or with the version of this construct in which putative NF-κB binding site (-394 to -384) has been deleted (n = 4-5 independent experiments per group). (i) Luciferase activity of HEK293 cells co-transfected with HDAC2 promoter—luciferase construct in combination with mock (pcDNA3.1), 0.05 and 0.1 μg of the Flag-p65 construct. The effect of Flag-p65 on HDAC2 promoter activity was prevented by co-transfection of proteasome degradation-resistant Flag-dn-IκBα (n = 4 independent experiments per group). (j,k) Representative immunohistochemical images of mouse frontal cortex injected with the AAV-CaMKIIα::mCherry-Cre construct. Note that signals of mCherry (red) co-localizes with CaMKIIα-positive (j), but not PV-positive (k), neurons. (l,m) Chronic clozapine treatment down-regulates protein levels of IκBα in the frontal cortex of HDAC2-cKO mice. Animals were treated chronically (21 days) with clozapine (10 mg/kg), or vehicle, and sacrificed one day after the last injection (n = 7 mice per experimental condition). Representative immunoblots are shown (l). Mean ± s.e.m. *P < 0.05; **P < 0.01; n.s., not significant. Two-way ANOVA with Bonferroni’s post-hoc test (h, P < 0.01, F1,13 = 14.06; i, P < 0.01, F1,18 = 8.05). Two-tailed unpaired t-test (l, P = 0.04, t12 = 2.18). Nuclei were stained in blue with DAPI (b,d,e,g,j,k). Scale bars, 20 μm (b,d,e,g,j,k).
