Figure 6.
PCDH19 regulates IEG expression and LSD1 alternative splicing
(A) RT-PCR in hippocampal neurons transduced with PCDH19 CTF (CTF-V5) or empty vector as control. The relative mRNA level of Nr4a1, c-Fos, Egr1, Cyr61, and Npas4 is shown (n = 3–4; ∗p < 0.05, Student’s t test). Data are presented as mean ± SEM.
(B) RT-PCR in hippocampal neurons transduced with PCDH19 shRNA or control shRNA (scramble). The relative mRNA level of Nr4a1, c-Fos, Egr1, Cyr61, and Npas4 is shown (n = 6–8; ∗p < 0.05, ∗∗p < 0.01, Student’s t test). Data are presented as mean ± SEM.
(C) Left panels, confocal images of hippocampal neurons transfected with control shRNA (scramble), CTF-V5 NLSBR1BR2, CTF-V5 NLSbr1BR2, PCDH19 shRNA (shRNA), and PCDH19 shRNA + PCDH19-V5 (shRNA + PCDH19, rescue condition) and treated with vehicle, NMDA, or DAPT (4 h) before immunolabeling for NR4A1, MAP2, and V5, as indicated. Scale bars, 40 μm. Bottom right panel, quantification of NR4A1 fluorescence intensity (entire neuron) (n, scramble 58, scramble + NMDA 32, CT-V5 NLSBR1BR2 + NMDA 20, CTF-V5 NLSbr1BR2 + NMDA 16, shRNA 35, scramble + DAPT 15, shRNA + PCDH19 17, shRNA + PCDH19 + DAPT 14; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA and Tukey’s post hoc test). Data are presented as mean ± SEM.
(D) Quantification of neuroLSD1 (nLSD1) isoform (%) in hippocampal neurons transduced as in (A) and (B) (n = 6, ∗∗∗p < 0.001, Student’s t test). Data are presented as mean ± SEM.
(E) Quantification of NOVA1 expression in western blots (top) and representative western blot (bottom) from hippocampal neurons transfected with control shRNA (scramble), PCDH19 shRNA (shRNA), and shRNA + PCDH19-V5 (rescue) (n = 3–4; ∗∗p < 0.01, one-way ANOVA and Dunnett’s post hoc test). Data are presented as mean ± SEM. Values are shown in Table S6.