Fig 4. KSHV LANA-mediated MCL-1 stabilization inhibits caspase-3-dependent apoptosis.

(A, left) TREx/BJAB cells stably expressing LANA-WT or LANA-P1 were stimulated with 1 μg/ml of Doxy, treated with etoposide (50 μM) for 24 h, and then assessed for Annexin V and propidium iodide (PI) staining by FACS analysis. Numbers indicate the percentage of cells in each quadrant. (A, right) The cell populations representing early (lower right quadrant) and late (upper right quadrant) apoptosis were summarized and shown as a graph. P-values were calculated using Student’s t-test (compared with the results for the T/BJAB-vector cell line). NS (Non-significant), p > 0.05; ***, p < 0.0005; N = 4. (B) Upon stimulation with Doxy (1 μg/ml), cells were also treated with etoposide (25 μM) for 24 h before harvesting. Cell lysates were then used for IB with an anti-caspase-3 antibody. (C) Cells were harvested after treatment with Doxy (1 μg/ml) and etoposide (25 μM) for 24 h, followed by treatment with MG132 (10 μM) for 6 h. Cell lysate were then used for IP with anti-FBW7 antibody and IB with either anti-Au or anti-MCL-1 antibodies.