MEF-2 inhibition perturbs HTLV-1-mediated T-cell transformation in correlation with a heightened MEF-2 expression in ATL patients. (A) PBMCs were transduced with scrambled or shMEF-2 expressing lentivirus by spinoculation. Viable cell proliferation of PBMCs was determined by trypan blue exclusion assay, after co-culture with lethally irradiated MT-2 cells for the indicated times. Error bars represent standard deviation of triplicate samples with high significance (***p < 0.001) between shMEF-2 versus shControl samples. (B) MT-2 and Jurkat cells were collected at 24 hr post a 3-day transfection followed by staining with propidium iodide (PI-25 μg/ml, RNAase- 40 μg/ml, sodium citrate-0.1% and Triton-100 × −0.03%). Cell cycle progression of MT-2 and Jurkat cells were observed via flow cytometry with no transfection (upper panel), mock transfected (middle panel) and shMEF2 plasmid (2.5 μg/1×106 cells) transfection (lower panel). The percentage of sub-G1 cells, G0/G1, S and G2-M cells was analyzed using the FLowjo software. (C) MEF-2A mRNA levels was determined by the quantitative real-time PCR as described in Methods. At least two replicates per donor were processed and MEF-2 levels were compared between seronegative controls and ATL patients (n = 3, each). Each point represents average mRNA expression in individual donors. Bars represent mean with Standard Error (SEM) derived by a two-tailed, unpaired nonparametric t-test (Mann–Whitney).