Figure 2

Dox promotes formation of DNA DSBs in primary neurons.

(A) Cortical neurons at 28–32 DIV were treated with a vehicle or with Dox (0.1 μM) or with DNA damaging drug etoposide (5 μM) overnight, fixed and stained for a marker of DSBs phosphorylated histone H2A variant X, γH2A.X (green), MAP2c (red) and with the nuclear Hoechst dye (blue) and imaged. The neuronal nucleus is enlarged on the Dox panel to illustrate the γH2A.X puncta. Note the green nuclear staining in cells treated with Dox and etoposide. Also note the reduced dendritic arborization in neurons treated with Dox and etoposide. Scale bar is 20 μm. (B) Images of fixed neurons treated with Dox (0.1 μm) overnight, Dox (0.01 μM) for 3 days, or etoposide (5 μM) overnight were analysed with an automated algorithm. The puncta index was estimated by measuring the standard deviation of γH2A.X fluorescence intensity. The puncta index of γH2A.X staining is increased in neurons treated with Dox and etoposide. ***p < 0.0001 (Dunnett’s test). A.u., arbitrary units. Several thousand neurons were analyzed. Results were pooled from at least three independent experiments. (C) Images of fixed neurons treated with Dox (0.1 μm) for indicated times were analysed for DNA DSBs. The levels of γH2A.X were estimated by measuring the γH2A.X fluorescence intensity. ***p < 0.0001 (Dunnett’s test). (D) An example of a neuron from cultures treated with Dox (0.1 μm) for 6 hours and stained as in (A). Note γH2A.X puncta in the nucleus. Scale bar is 5 μm.