TCL1A overexpression leads to aberrant cell cycle progression. (A) Immunoblots of co-IPs of B cells from spleens of WT or Eμ-TCL1A mice using anti-TCL1A antibodies or isotype IgG (negative control). Lanes represent individual animals. (B) Immunoblots of co-IPs using IgG controls or specific TCL1A antibodies in primary CLL samples cocultured with differentiated THP-1 monocytic cells in the presence of CpG and IL-15 for 36 hours to induce proliferation. Lanes represent individual CLL samples. (C) Box plot showing percentages of cells in G0/G1 (top) and G2/M (bottom) of JVM3 ± TCL1A that were released from RO-3306 synchronization from 4 independent experiments. Boxes display medians with 25th to 75th percentiles and whiskers of 5th to 95th percentiles. Significances were estimated by the Mann-Whitney test. Analogous results for HEK293T and DoHH2 cells (TCL1A introduction) as well as for MEC1 cells (short hairpin RNA (shRNA)–mediated TCL1A knockdown) are shown in supplemental Figure 6A. (D) Flow-cytometric analysis of G2/M synchronized (via RO-3306 for 20 hours) JVM3 cells ± TCL1A stained with Hoechst. Representative histogram shows an accelerated exit from the G2/M into the G1 cell cycle phase after release in full medium of TCL1A-overexpressing cells. The blue area indicates JVM3 empty vector (EV) and red area indicates JVM3^TCL1A. (E) Representative immunoblot from lysates of cells released from RO-3306–mediated synchronization. JVM3^TCL1A cells show faster dephosphorylation of proteins involved in the regulation of cell cycle transition as well as a faster degradation of cyclin A and B and CDC20. Analogous results for HEK293T and DoHH2 cells (TCL1A introduction) as well as for MEC1 cells (TCL1A knockdown) are shown in supplemental Figure 6B. (F) Aberrant cell cycle progression in TCL1A-driven murine leukemia. Eμ-TCL1A mice (N = 4) or age-matched WT controls (N = 3) were injected with 5-bromo-2′-deoxyuridine (BrdU) (top). At 20 hours after injection, the ratios of BrdU⁺ (in S phase at time of injection, green), phosphorylated histone 3⁺ (pH3⁺) (in mitosis at time of harvest, red), BrdU⁺;pH3⁺ (formerly in S phase, currently mitotic, yellow), and BrdU⁻;pH3⁻ (not proliferating, blue) cell counts were determined by immunostaining (2500 cells per animal; Student t test). Images were captured at original magnification ×60 using an Axio Scope.A1 fluorescent microscope. Increased overall proliferation (S phase and mitosis) of Eμ-TCL1A cells (BrdU⁺ and pH3⁺) (bottom). These TCL1A-transgenic cells exited the cell cycle more often (higher proportion of BrdU⁺;pH3⁻ and similar proportion of BrdU⁺/pH3⁺ cells). An increased population of BrdU⁻/pH3⁺ cells in the Eμ-TCL1A cohort also suggests that they re-enter the cell cycle more frequently.