Species-specific disruption of PML-NBs in cells stably expressing IE1.

(A, B) Effect of humIE1 and ratIE1 on the integrity of PML foci in human fibroblasts. Human fibroblasts with doxycycline-inducible expression of FLAG-tagged humIE1 (HFF/humIE1), FLAG-tagged ratIE1 (HFF/ratIE1) or control cells (HFF/control) were either left untreated (- Dox) or were treated with doxycycline (+ Dox) for 24 h. The cells were fixed for immunofluorescence staining of endogenous humPML and of IE1 proteins using an anti-FLAG antibody (A), followed by quantitation of humPML foci numbers in 50 cell nuclei per sample (B). (C) Impact of humIE1 and ratIE1 on the SUMOylation state of humPML. HFF/humIE1, HFF/ratIE1 or control cells were either left untreated (- Dox) or were treated with doxycycline (+ Dox). 24 h later, cells were harvested for Western Blot detection of IE1 proteins using an anti-FLAG antibody (upper panel), humPML (middle panel), and β-actin as loading control (lower panel). (D, E) Effect of humIE1 and ratIE1 on the integrity of PML foci in rat fibroblasts. Rat fibroblasts with doxycycline-inducible expression of FLAG-tagged humIE1 (REF/humIE1), FLAG-tagged rIE1 (REF/ratIE1) or control cells (REF/control) were either mock treated (- Dox) or were treated with doxycycline (+ Dox) for 24 h. The cells were fixed for immunofluorescence staining of endogenous ratPML and for IE1 proteins using an anti-FLAG antibody (D), followed by quantitation of ratPML foci numbers in 50 cell nuclei per sample (E). (F) Impact of humIE1 and ratIE1 on the SUMOylation state of ratPML. REF/humIE1, REF/ratIE1 or control REF were either left untreated (- Dox) or were treated with doxycycline (+ Dox). 24 h later, cells were harvested for Western Blot detection of IE1 proteins using an anti-FLAG antibody (upper panel), ratPML (middle panel), and β-actin as loading control (lower panel).