Fig. 4.

Evaluation of γH2A.X and H2A.X levels in extracellular tachyzoites. (A) Extracellular tachyzoites of RHΔHXGPRT or RHΔKU80 strains were treated with DMSO 0.1%, topotecan (50 μM), HCPT (12 μM) or phleomycin (50 μM) at 37 °C for 4 h. Phleomycin was used as a positive control for the generation of extracellular damage. Subsequently, tachyzoites were washed, fixed with 4% PAF, and IFA was performed. Anti-SAG1 mouse, anti-H2A.X rabbit and anti-γH2A.X rabbit were used as primary antibodies. The figure shows one representative image obtained for each antibody H2A.X and γH2A.X (red) and Sag1 (green). γH2A.X mark was not detected with topotecan and HCPT drugs, as well as DMSO (image shown) but we obtain positive marks in parasites treated with phleomycin (phleo). However, in all conditions H2A.X signal was observed. Scale bar 5 μM. (B) H2A.X and γH2A.X positive tachyzoites were quantified under different conditions and plotted using GraphPad software.