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. 2014 Oct 23;10(10):e1004458. doi: 10.1371/journal.ppat.1004458

Figure 6. TRAF6 and MCL-1 are required for ATL cell survival and HTLV-1-mediated T-cell immortalization.

Figure 6

(A and B) Requirement of TRAF6 and MCL-1 for the viability of HTLV-1 transformed and ATL cell lines. (A) Cell viability assay was performed at 6 days after TL-OM1 and MT-2 cells were transduced with lentiviruses expressing the indicated shRNAs. Relative cell viability (%) was expressed as a percentage relative to the control cells. (B) Flow cytometric analysis of ATL cell lines transduced with shRNAs as described in (A). Cells were stained with both annexin-V-Alexa Fluor 488 and propidium iodide (PI). The distribution of cells is indicated as a percentage in each quadrant. (C) MCL-1 protein is overexpressed in HTLV-1-transformed T cells. Immunoblotting was performed with whole cell lysates of primary human CD4+ T cells (0 W), CD4+ T cells immortalized with HTLV-1 by co-culture with irradiated MT-2 cells for 12 weeks (12 W), Jurkat, ATL (TL-OM1) and HTLV-1 transformed cell lines (MT-2 and C8166). (D) TRAF6 and MCL-1 are essential for the immortalization of T cells by HTLV-1. At four and six weeks after the co-culture of shRNA-transduced PBMCs and irradiated MT-2 cells, viable cells were counted using the trypan blue exclusion method. Puromycin (5 µg/ml) was added at 4 weeks after the co-culture. The data are representative of two independent experiments.