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Research Paper Volume 9, Issue 9 pp 1957—1970

The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons

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Figure 4. Pyridostatin downregulates BRCA1 levels in primary neurons. (A) A scheme of the Brca1 gene and its promoter showing putative G-quadruplex sequences. (B) Primary neurons were treated with a vehicle (control, -) or with different concentrations of pyridostatin (PDS; 1, 2 or 5 μM) overnight. Then neurons were collected, lysed, and samples were processed with western blotting. Note that pyridostatin reduced BRCA1 protein levels in a dose-dependent manner. Actin was used as a loading control. (C) Quantification of BRCA1 protein levels normalized to actin from (B). **p(cont-1 μM)=0.005, **p(cont-2 μΜ)=0.0015, **p(cont-5 μΜ)=0.0043 (ANOVA). N.s., non-significant, p(1 μΜ−2 μΜ)=0.0547, p(1 μΜ−5 μΜ)=0.0854. Results were pooled from five independent experiments. (D) Primary neurons were treated with a vehicle (control, -) or with a G-quadruplex stabilizing drug, TmPyP4 (1 μM), overnight. Neurons were lysed, and samples were processed by western blotting. TmPyP4 reduced BRCA1 protein levels. Actin was used as a loading control. (E) Quantification of BRCA1 protein normalized to actin from (D). *p<0.0276 (t-test). Results were pooled from four independent experiments. (F) Primary neurons were treated with a vehicle (control; -) or with 2 μM pyridostatin (PDS; +) overnight. The expression of Brca1 was determined by qRT-PCR relative to Actin. *p=0.0445 (t-test). Results were pooled from three independent experiments. (G) Primary neurons were treated with a vehicle (control; -) or with 2 μM pyridostatin (PDS; +) overnight. Relative expression of the Actin, Gapdh and Tbp genes was determined by qRT-PCR. *p=0.0112, **p=0.0039 (t-test). N.s., non-significant (p=0.7583). Results were pooled from three independent experiments. (H) Primary neurons were treated with a vehicle (control; -) or with 2 μM pyridostatin (PDS; +) overnight. The expression of Brca1 was determined by qRT-PCR normalized to Tbp. **p=0.0033 (t-test). Results were pooled from three independent reactions.