Brd4 Is Displaced from HPV Replication Factories as They Expand and Amplify Viral DNA
Figure 2
Salt extraction of cells expressing HPV16 E1 and E2 show that the E1 protein binds most tightly to host nuclei.
Human keratinocytes were cotransfected with expression vectors for HPV16 E1 (pMEP9-E1), HPV16 E2 (pMEP4-E2), or both proteins. Cells were either fixed directly in paraformaldehyde (PFA), or proteins were extracted from cells in buffers containing either 100 mM or 300 mM NaCl prior to fixation in PFA. A. E1, E2 and endogenous Brd4 proteins were detected by indirect immunofluorescence. Staining for the E1 protein is shown in green, the E2 protein in cyan, and Brd4 in red. The dashed blue lines represent the perimeter of the nuclei (identified by DAPI staining, not shown). B. Level of E1, E2, and Brd4 were measured in individual cells extracted and fixed as described above using the Contour feature of Imaris software (Bitplane) to extract values from each channel in individual nuclei. Average values obtained from at least five reconstructed nuclei are shown and the error bars represent the standard deviation. Individual transfected cells could not be identified in cells transfected with the empty pMEP4/9 vectors or in cells transfected with HPV16 E2 vectors and extracted in 300 mM NaCl containing buffer. In these samples, random cells were selected for quantitation.