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. 2016 May 27;11(5):e0156439. doi: 10.1371/journal.pone.0156439

Fig 5. TOP1 is necessary but not sufficient to unsilence Ube3a.

Fig 5

(A) WT cortical neuron cultures were transfected with tdTomato and Cas9 alone (Ctrl.) or Cas9 and a sgRNA directed to Top1 at DIV 3. Neurons were then treated with vehicle (DMSO) or 300 nM topotecan for 72 hours. Scale bar, 20 μm. (B) Quantification of TOP1 (top) or UBE3A (bottom) fluorescence. Values were normalized to the fluorescence intensity of control neurons. Mean ± s.e.m., unpaired student’s t-test; * p < 0.05, n = 3 cultures. (C) Ube3am-/p+ (AS) cortical neuron cultures were transfected with tdTomato and Cas9 alone (Ctrl.) or Cas9 and a sgRNA directed to Top1 at DIV 3. Neurons were then treated with vehicle (DMSO) or 300 nM topotecan for 72 hours. Scale bar, 20 μm. (D) Quantification of TOP1 (top) or UBE3A (bottom) fluorescence. Values represent raw integrated density values divided by a value of 1000. Mean ± s.e.m., unpaired student’s t-test relative to vehicle-treated Ctrl.; * p < 0.05, n = 3 cultures. N.D. = Not detected.