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. 2020 May 11;295(25):8613–8627. doi: 10.1074/jbc.RA119.010266

Figure 3.

Figure 3.

GluN3A knockdown enhances MEF2C-dependent transcription. A, Luciferase expression in hippocampal neurons transfected with MRE-Luc plasmid and infected with either Grin3a shRNA1 or control (Ctrl) lentiviruses. Neurons were stimulated with TTX w/d for the indicated amounts of time prior to lysis. Data are expressed as fold induction over TTX (0 h). Two-way ANOVA for time, F(4,62) = 34.65, p < 0.0001; for virus, F(1, 62) = 32.16, p < 0.0001; and for virus–time interaction, F(4, 62) = 7.602, p < 0.001. n = 11 at 0 h, 6 at 4 h, 7 at 6 h, 7 at 8 h, and 6 at 22 h. Grin3a shRNA versus Ctrl at 6 h and 8 h, p < 0.0001. B, Luciferase expression in hippocampal neurons transfected with CRE-Luc plasmid and infected with either Grin3a shRNA1 or Ctrl lentiviruses. Neurons were stimulated as described for panel A. Two-way ANOVA for time, F(5, 24) = 23.00, p < 0.0001; virus, F(1, 24) = 1.407, p = 0.2471. n = 4 at 0 h, 4 at 4 h, 2 at 6 h, 4 at 8 h, 2 at 14 h, and 2 at 22 h. C, Levels of Bdnf IV mRNA in hippocampal neurons infected both with lentiviruses containing Grin3a shRNA1 or its paired control and lentiviruses expressing shRNAs targeting individual MEF2 family members. Two-way ANOVA for treatment, F(1, 77) = 68.53, p < 0.001; for virus, F(7, 77) = 2.520, p = 0.022; and for treatment–virus interaction, F(7, 77) = 2.254, p = 0.039. n = 6/condition, except Ctrl TTX, n = 4, and Mef2c+Grin3a double KD TTX w/d, n = 5. For each viral condition, TTX w/d was significantly different from TTX, and for TTX, there were no significant differences between viral conditions. Significant TTX w/d post hoc comparisons: Ctrl versus Grin3a KD, p = 0.0093; Grin3a KD versus Mef2c+Grin3a double KD, p = 0.041. D, Luciferase expression in hippocampal neurons cotransfected with the pUAS-Luc plasmid, expression vectors for Gal4 fusions of the indicated MEF2C splice variants, and Grin3a shRNA1 or its paired Ctrl1 vector. Data are shown as relative luciferase expression compared with control conditions (Ctrl1 and TTX). Two-way ANOVA for vector, F(2, 31) = 12.67, p < 0.0001; for virus/treatment, F(3, 31) = 6.803, p = 0.0012; and for vector–virus/treatment interaction, F(6, 31) = 3.318, p = 0.012. For Gal4-MEF2Cα1β, Grin3a KD TTX versus TTX w/d, p = 0.0003; Grin3a KD TTX w/d versus Ctrl TTX w/d, p = 0.0027, n = 4 for Ctrl TTX w/d and Grin3a KD TTX and 3 for Grin3a KD TTX w/d. E and F, Levels of TTX w/d-induced Bdnf IV mRNA (E) or Arc mRNA (F) in hippocampal neurons infected with the indicated lentiviruses. Arc mRNA was measured following 30 min TTX w/d. Induced mRNA levels are shown as percentages of induction relative to control conditions (Ctrl). Bdnf IV ANOVA for virus, F(3, 20) = 25.85, p < 0.0001, n = 6/sample; Ctrl versus KD, p < 0.0001; KD versus dKD, p = 0.0007; dKD versus rescue, p = 0.0002. Arc ANOVA for virus, F(3, 19) = 9.641, p = 0.0004. n = 6/sample, except double KD, n = 5. Ctrl versus KD, p = 0.0066; KD versus dKD, p = 0.0024; double KD versus rescue, p = 0.006. *, p < 0.05 for Grin3a KD versus Ctrl; #, p < 0.05 for dKD versus Grin3a KD; ∧, p < 0.05 for rescue versus dKD.