Supplementary Figure 9: Effect of activation of 5-HT2A receptor on IκBα mRNA expression in Flp-In T-REx HEK293 cells
From: Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects
(a,b) Generation of Flp-In T-REx HEK293 cells that express c-Myc-5-HT2A-eCFP or c-Myc-5-HT2A-I181D-eCFP in an inducible manner. These cells were maintained in the presence of 10 ng/ml doxycycline for 24 h, and then imaged to detect eCFP (a). Membranes were prepared from such cells, and density of c-Myc-5-HT2A-eCFP and c-Myc-5-HT2A-I181D-eCFP was measured by radioligand binding assays with [3H]ketanserin (n = 2 independent experiments per group; b). (c) Time-course of the effect of 5-HT (10 μM) on expression changes of IκBα mRNA in Flp-In T-REx HEK293 cells that harbored the human c-Myc-5-HT2A-eCFP receptor (n = 3 independent experiments per group). (d) The effect of 5-HT on IκBα mRNA expression is blocked by the 5-HT2A antagonist M100907. Cells were treated for 60 min with 5-HT (10 μM) or vehicle after being pre-treated for 15 min with M100907 (10 μM) or vehicle (n = 2-3 independent experiments per group). (e) The effect of 5-HT (10 μM) on IκBα mRNA expression is absent in Flp-In T-REx HEK293 cells harboring c-Myc-5-HT2A-I181D-eCFP at the Flp-In locus—a mutant receptor that does not activate Gq/11 proteins19 (n = 3 independent experiments per group). (f) The effect of 5-HT (10 μM) on IκBα mRNA expression is blocked by the MEK1/2 inhibitor SL-327(10 μM) in Flp-In T-REx HEK293 cells that harbored the human c-Myc-5-HT2A-eCFP receptor at the inducible Flp-In locus (n = 3 independent experiments per group). (g) Chronic treatment with clozapine (10 mg/kg), risperidone (4 mg/kg), quetiapine (10 mg/kg), sulpiride (10 mg/kg), or volinanserin (1 mg/kg), but not with haloperidol (1 mg/kg), down-regulates 5-HT2A receptor density in mouse frontal cortex (see also Fig. 5h). Mice were treated chronically (21 days) with the indicated drug, or vehicle, and sacrificed 24 h after the last injection. [3H]Ketanserin binding was tested in frontal cortex plasma membrane preparations (n = 6 mice per experimental condition; see also Fig. 5h). Mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Extra sum-of-squares test (b, P = 0.53, F1,26 = 0.39). One-way ANOVA with Bonferroni’s post-hoc test (c, P < 0.001, F3,8 = 16.20; d, P < 0.01, F3,7 = 10.20; f, P < 0.001, F3,8 = 139.3). Two-tailed unpaired t-test (e, P = 0.52, t4 = 0.70).
