FIG 9.

RNA Pol II Ser 5, Brd4, and acetylated histones are localized predominantly inside the replication foci in HPV16 ΔE8^E2 cells. (A) HFK (39 strains) cell lines containing HPV16 wild-type or HPV16ΔE8^E2 genomes were immunostained with antibodies against macroH2A1.2 (green), Brd4 (red), and RNA Pol II Ser 5 (purple). Nuclei were stained with DAPI (indicated by white dotted line). Quantitation from two independent experiments is shown to the right. In the upper graph, in 12 HPV16 wild-type cells, 100% of foci (13/13) showed enrichment of mH2A1.2. In 32 HPV16 ΔE8^E2 cells, 192 RPA-positive foci were scored for either depletion or a small residual amount of macroH2A1.2 at the core. Foci indicated with a white arrow show the core staining of macroH2A1.2. In the lower graph, the locations of RNA Pol II Ser 5 and Brd4 with respect to the foci are shown from HPV16 wild-type cells (13 foci) and HPV16ΔE8^E2 cells (192 foci in 32 cells). Foci were visually counted from two biological experiments. (B) HPV16 wild-type and HPV16 ΔE8^E2 genome-containing cells were immunostained with antibodies against histone H3K9/18 acetyl (purple), macroH2A1.2 (green), and RPA (red). Nuclei were stained with DAPI (not shown), and the distributions of H3K9/18 acetyl in HPV16 wild-type (n = 35, 37 RPA-positive foci) and HPV16ΔE8^E2 cells (n = 44, 188 RPA-positive foci) were analyzed by visual counting. Cells were scored from two biological independent replicates. (Lower panel) Fluorescence intensity scans obtained by drawing a line through a nucleus using Leica LAS X software. A gray bar above the scan delineates the position of the replication focus.