Figure 2. Pyridostatin promotes the formation of 53BP1-positive puncta in primary neurons. (A) Primary cortical neurons were transfected with GFP and mApple-53BP1trunc constructs, and then treated with a vehicle (left panel; control) or with 1 μM pyridostatin (right panel; PDS). Neurons were imaged with an automated microscope every 24 h for 3 days. Scale bar is 5 μm. (B) Quantification of the mApple-53BP1trunc puncta index from (A) at different times. The puncta index was estimated by measuring the standard deviation of the 53BP1 fluorescence intensity. Note that 53BP1 puncta index is higher in pyridostatin-treated neurons than control neurons. *p<0.01, **p<0.001, and ***p<0.0001 (t-test). A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments. (C) Primary cortical neurons were treated with a vehicle (upper panel; control) or with 1 μM pyridostatin (bottom panel; PDS) overnight, fixed, and stained for MAP2c (red), a marker of DNA damage 53BP1 (green), and with the nuclear Hoechst dye (blue). Scale bar is 10 μm. (D) Quantification of the 53BP1 puncta index from (C). Pyridostatin (PDS) increased the 53BP1 puncta index compared to control neurons (cont). ***p<0.0001 (t-test). A.u., arbitrary units. Three hundred neurons were analyzed from three independent experiments.