Increased expression of the cell cycle marker phosphohistone H2Ax in Wlds transfected HEK293 cells. Confocal micrographs of HEK293 cells 5 days after transfection with either an (a-f) enhanced green fluorescent protein (eGFP)-Wldsconstruct 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 Wldsis being expressed. Note that not all cells have transfected with construct, and non-Wldsexpressing cells identifiable by their TOPRO3 labeled nuclei do not have corresponding H2Ax puncta. H2Ax puncta were found in all Wlds-expressing cells, regardless of the nuclear distribution of Wlds(panels a to c show Wldsin nuclear inclusions; panels d to f show Wldsexpressed in a strong diffuse manner throughout the nucleus). Scale bar 10 μm.