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. Author manuscript; available in PMC: 2016 Dec 2.
Published in final edited form as: Neuron. 2015 Dec 2;88(5):902–909. doi: 10.1016/j.neuron.2015.11.018

Figure 3. Silencing of C9ORF72 in C9BAC mice and primary cortical neurons.

Figure 3

(A) Sense and antisense foci are detected in cultured cortical neurons of C9BAC embryos at 10 days in vitro (DIV). These foci were not visible following RNase treatment and were not detected in Ntg neurons, or by probes targeting the myotonic dystrophy associated type-1 (CTG)n or type-2 (CCTG)n expansions (DM1 and DM2 respectively, scale bar = 5 μm). (B). Concentration of poly(GP) in cultured cortical neurons derived from individual C9BAC or Ntg littermate embryos measured by ELISA (mean±SEM, unpaired t-test, Ntg n=2, C9BAC n=6, unpaired t-test). (C) No significant differences were detected in the response of cultured cortical neurons derived from individual C9BAC and Ntg embryos to the autophagy inhibitors chloroquine or 3-methyladenine (two independed experiments, individual embryos cultured; C9BAC n=6, Ntg n=5, mean±SEM, 2-way ANOVA, Bonferroni's multiple comparison, * p<0.05). (D) ddPCR analysis of all transcripts of C9ORF72 (Vall) relative to HPRT in two independent experiments (Exp. 1. individual embryos n=4, mean±SEM, one-way ANOVA, Bonferroni's multiple comparison test, * p<0.05, *** p<0.0001; Exp. 2. mixed embryo culture, n=3, one-way ANOVA, Bonferroni's multiple comparison test *** p<0.001). (E) Levels of the mature microRNA product (normalized to snoRNA135) of rAAV-GFP-miRC9 correlated with percent silencing of C9ORF72 relative to rAAV-GFP from Exp. 1 (black) and Exp. 2 (blue). (F) Levels of poly(GP) quantified by ELISA from primary cortical neuron cultured derived from two independent experiments (Exp. 1. individual embryos n=2, mean±SEM, one-way ANOVA; Exp. 2. mixed embryo culture, n=3, one-way ANOVA, Bonferroni's multiple comparison test, ** p<0.001).