Fig. 4: (S)-PHA533533 unsilences paternal Ube3a via a mechanism that is independent of CDK2/CDK5 and topoisomerase 1 (TOP1) inhibition.

Quantification and representative western blots showing that ASO-mediated knock-down of (a, b) CDK2, (c, d) CDK5, or (e, f) TOP1 failed to unsilence paternal Ube3a-YFP or occlude the unsilencing of paternal Ube3a-YFP by (S)-PHA533533 in primary cortical neurons, whereas (e, f) ASO-mediated knock-down of TOP1 occluded the TOP1 inhibitor topotecan from unsilencing paternal Ube3a-YFP. For these experiments, mouse primary neurons derived from wild-type or paternal Ube3a-YFP reporter mice (Ube3a^m+/pYFP) were treated as indicated with 10 µM active or scrambled (Scr) ASOs at DIV5 followed by treatment with 0.1% DMSO (vehicle control), 1 µM (S)-PHA533533, or 0.3 µM topotecan for 72 h starting at DIV7 (n = 3 experiments; mean ± SEM). g TOP1-based DNA relaxation assay verified that (S)-PHA533533 and (R)-PHA533533 do not target TOP1. The addition of TOP1 catalyzes supercoiled DNA substrate (lane 1) into slower migrating relaxed DNA (lane 2). The presence of different concentrations of (S)-PHA533533 (lanes 3–5) or (R)-PHA533533 (lanes 6–8) did not result in an increased amount of uncut supercoiled DNA compared to DMSO control (lane 2), whereas the presence of 3 µM topotecan did (lane 9). Data shown are representative of multiple independent trials, all confirming similar outcomes. h–j (S)-PHA533533 reduced the expression of Ube3a-ATS, but not other long genes or other antisense transcripts. Following the treatment, the relative quantities of (h) Snord115, (i) Dlg2, (j) Nrxn3, (k) Malat1, (l) Pantr1, and (m) Airn transcripts were determined by quantitative RT-PCR (n = 4 experiments; mean ± SEM). All data in (a–f) and (h–m) were normalized to GAPDH/Gapdh or β-TUBULIN expression, log-transformed, mean-centered, and autoscaled for statistical analysis (one-way ANOVA followed by Tukey’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS non-significant). μM micromolar, DMSO dimethyl sulfoxide, kDa kilodalton, patUBE3A-YFP paternal UBE3A fused to yellow fluorescent protein, YFP yellow fluorescent protein, WT wild-type. Source data and comprehensive statistics, including F-values, degrees of freedom, confidence intervals, and exact p values, are provided as a Source Data file.