Fig. 3. UBE3A expression is recovered in neurons derived from corrected AS iPSCs.
(A) Representative immunofluorescence images of neurons derived from WT, AS, and AS neo-ex HR iPSCs for neuron-specific marker class III β-tubulin (TUJ1, red) and the dopaminergic neuron-specific marker tyrosine hydroxylase (TH, green). Nuclei were counterstained blue with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 50 μm. (B) Bisulfite sequencing analysis of SNURF/SNRPN CGI in DA neurons. The primers for bisulfite PCR are shown in fig. S8A. Percentage of DNA methylation and P value (versus WTand versus AS) are shown at the bottom. Filled and open circles represent methylated and unmethylated CpGs, respectively. Statistical significance was determined by unpaired Student’s t test. (C) Quantitative RT-PCR analysis of UBE3A antisense RNA expression normalized to GAPDH mRNA in DA neurons. Error bars indicate ±SEM of three independent experiments. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple-comparison test (versus AS DA, **P < 0.005). (D and E) Western blot analysis of total cell lysates from iPSCs and DA neurons with anti-UBE3A monoclonal and polyclonal antibodies, an anti–β-actin antibody (D), and an anti–α-synuclein antibody (E).