Forced expression of HBZ promotes G1/S transition and apoptosis in primary CD4+ T cells. (a, b) Mouse primary CD4+ T cells were transduced with HBZ and cultured in low IL-2 (10 U/ml) containing medium. Thirty-six hours after IL-2 withdrawal, transduced CD4+ T cells were purified using a FACSAria2 and cultured in fresh medium containing higher IL-2 (40 U/ml). Cell cycle distribution was monitored at the indicated times (0, 18, 24 and 30 h after release from G1 phase). Three independent experiments were performed in triplicate, and the representative data are shown. (c) GFP competition assay was carried out using bicistronic GFP expression vectors. After retroviral transduction of each construct, the proportion of transduced cells (GFP+ fraction) was measured by flow cytometry at the indicated date. Three independent experiments were performed in triplicate, and the representative data are shown. (d) Relative expression levels of cell cycle inducing genes and (e) proapoptotic genes were evaluated by qPCR. The expression level of genes was measured in triplicate. Statistical significance was determined by the two-tailed paired Student's t-test. N.D., not detectable. Asterisks indicate the statistical significance as follows: *P < 0.05; **P < 0.01; ***P < 0.001.