Tax overactivates replication origins. (A-B) JPX9 and JPX9i cells were extracted with CSK buffer, fixed, and stained for MCM2 or MCM3 and DNA content (4′,6-diamidino-2-phenylindole) before flow cytometric analysis. (A) Representative analyses of MCM2 staining and DNA content labeling. (B) MCM2 and MCM3 MFI in G1 and M phases of the cell cycle normalized to the MFI of control cells in G1 (relative MFI). (C) JPX9 and JPX9i were treated with HU (2.5mM) to synchronize cells at G1/S transition for 16 hours, and MCM2 occupancy at Myc, LaminB2, β-globin, and RNF185 replication origins was detected by ChIP. Results were expressed as a percentage of input DNA. (B) Representative results of one of 3 independent ChIP experiments are shown. (D) HeLa cells were transfected with a Tax expression vector, incubated during 3 hours, and synchronized at G1/S transition with 2mM HU. After a short BrdU pulse (15 minutes) in HU-free medium, cells were processed for confocal microscopy. (E) Number of BrdU foci, (F) BrdU foci intensity, and (G) integrated BrdU intensity in control (Ctrl) and Tax-positive cells (n = 50). (H-I) Rat-1 cells stably transduced with control (Ctrl) or Tax-expressing viruses were treated or not with UCN-01 (100nM, 3 hours), pulsed with BrdU (33μM, 30 minutes). DNA was isolated, spread on slides, and the BrdU-labeled tracks were analyzed by confocal microscopy. Next, fork-to-fork distance and length of isolated tracks were measured. (J-K) JPX9 cells were stably transduced with lentiviruses encoding MCM3 shRNAs, stimulated (black bars) or not (open bars) with ZnCl2 (120μM), and pulsed with BrdU for 30 minutes. (J) MFI of BrdU labeling and (K) duration of S phase were analyzed by flow cytometry (see supplemental Figure 4 for details). The results presented are the mean ± SD of 3 independent experiments. A.U. indicates arbitrary units; and NS, not significant. *P < .05, according to Kruskal-Wallis test. ***P < .001, according to Kruskal-Wallis test.
