Figure 5

Increased expression of the cell cycle marker phosphohistone H2Ax in Wld^s transfected HEK293 cells. Confocal micrographs of HEK293 cells 5 days after transfection with either an (a-f) enhanced green fluorescent protein (eGFP)-Wld^sconstruct or (g-i) a eGFP-only control construct. Immunocytochemical labeling of phosphohistone H2Ax is shown in red, the nuclear marker TOPRO3 is shown in blue, and constructs are expressing in green (panels a, d and g show H2Ax and TOPRO3; panels b, e and h show construct and TOPRO3; and panels c, f and i show all three markers). Note how phosphohistone H2Ax protein can only be seen in nuclear puncta where Wld^sis being expressed. Note that not all cells have transfected with construct, and non-Wld^sexpressing cells identifiable by their TOPRO3 labeled nuclei do not have corresponding H2Ax puncta. H2Ax puncta were found in all Wld^s-expressing cells, regardless of the nuclear distribution of Wld^s(panels a to c show Wld^sin nuclear inclusions; panels d to f show Wld^sexpressed in a strong diffuse manner throughout the nucleus). Scale bar 10 μm.