Figure 3.

HBZ reduces H3K18ac in cells. (A) The level of H3K18ac is reduced in HeLa cells expressing HBZ compared to control cells. HeLa cell lines carrying the empty or the HBZ expression vector, as indicated, were analyzed by indirect immunofluorescence confocal microscopy (magnification 40×) to compare levels of H3K18ac. (B) A graphical representation of immunofluorescence data. The mean signal density per cell for H3K18ac or H3 was determined for HBZ-expressing or control cells. Data are the average values (± S.E.M.) obtained from >100 cells. The asterisk denotes a P-value <0.0001 for a two-tailed Student t-test. (C) Jurkat cell lines were established that express HBZ with Myc and 6×His epitope tags or carry the empty vector. HBZ was immunoprecipitated from whole-cell extracts using an antibody against the 6×His tag and then detected by western blot using an antibody against the Myc tag. (D) HBZ is localized to the nucleus in Jurkat cells stably expressing HBZ. The indicated Jurkat cells were analyzed by indirect immunofluorescence confocal microscopy (magnification ×100/zoom × 2) for HBZ expression. NUP98 was stained to mark the nuclear membrane. (E) The level of H3K18ac is reduced in Jurkat cells expressing HBZ compared to control cells. The indicated Jurkat cell lines were analyzed by immunofluorescence confocal microscopy (magnification ×100/zoom × 2) to compare levels of H3K18ac. (F) Histones extracted from HeLa and Jurkat cells stably expressing HBZ exhibit less H3K18ac than control cells. Histones were resolved by SDS–PAGE and analyzed by western blot using the antibodies indicated. (G) Transient expression of HBZ reduces H3K18ac. Histones (2 µg) from HeLa cells transfected with E1A, HBZ or the empty pcDNA vector were analyzed by western blot with the antibodies indicated.