(A) (Left panel): KMS-11 cells were treated with TAK-580 (0–20 μM) in combination with LEN (0–20 μM) for 48 h. In each case, the cell viability of triplicate cultures was assessed with the Cell Titer-Glo® Cell Viability Assay and expressed as the percentage of the untreated control. Data are the mean ± SD. (B): Isobologram analysis shows the synergistic or additive cytotoxic effect of TAK-580 and LEN in Figure 5A. Light blue, orange, red, and blue rhombuses indicate CI values of the combination of TAK-580 and LEN; 0.63 (TAK-580 10 μM and LEN 5 μM), 0.62 (TAK-580 10 μM and LEN 20 μM), 0.63 (TAK-580 20 μM and LEN 5 μM), and 0.56 (TAK-580 20 μM and LEN 20 μM), respectively. CI < 1.0 indicates synergism; CI = 1.0 indicates an additive effect; and CI > 1.0 indicates antagonism. CI, combination index; Fa, fraction affected. (C) KMS-11 cells were cultured with TAK-580 (20 μM), LEN (40 μM), or TAK-580 plus LEN for 72 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Apoptosis was assessed as the percentage of annexin V-positive cells. (D) (Left panel): KMS-11 cells were treated with TAK-580 (20 μM) alone or in combination with LEN (20 μM) for 48 h. Whole-cell lysates were subjected to western blotting using c-Myc and β-Actin Abs. (Right panel): The graph represents ratios of c-Myc density relative to β-Actin in Figure 5D.