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

Figure 3. G-quadruplex-stabilizing drugs upregulate γH2A.X. (A) Primary cortical neurons were treated with a vehicle (control) or with 1 μM pyridostatin (PDS) or with 200 nM bortezomib (bortezomib) or with 10 μM cisplatin (cisplatin) overnight, fixed, and stained for MAP2c (red), a marker of DNA DSBs phosphorylated histone H2A variant X, γH2A.X (green), and with the nuclear Hoechst dye (blue). Scale bar is 10 μm. (B) The puncta index was estimated by measuring the standard deviation of the γH2A.X fluorescence intensity. Primary cortical neurons were treated with a vehicle (control, cont) or with 1 μM pyridostatin (PDS), or with 200 nM bortezomib (bort), or with 10 μM cisplatin (cisp) overnight, then fixed, and immunostained with antibodies against MAP2c and γH2A.X, and co-stained with the nuclear Hoechst dye (blue). ***p<0.0001 (t-test). N.s., non-significant (pbort=0.1995; pcis=0.8228). A.u., arbitrary units. Four hundred neurons were analyzed from three independent experiments. (C) Primary cortical neurons were treated with a vehicle (control, cont) or with a G-quadruplex stabilizing drug, TmPyP4 (1 μM, overnight), then fixed, immunostained against MAP2c and γH2A.X, and co-stained with the nuclear Hoechst dye (blue). ***p<0.0001 (t-test). A.u., arbitrary units. Four hundred neurons were analyzed from three independent experiments.