Figure 4

Amuvatinib inhibits growth, induces cell cycle arrest, and inhibits DNA synthesis in myeloma cell line U266. (A) U266 cells were treated with amuvatinib at various concentrations for 24 (■), 48 (▲), and 72 h (▼). Cells were counted using a Coulter counter, and the effect of the treatment on cell growth was determined. The data are presented in a time- and dose-dependent manner as percentages of time-matched controls. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05. (B) To determine the effect of amuvatinib on the cell cycle, U266 cells were treated with 3 or 5 μM amuvatinib or DMSO for 72 h. Flow cytometry was used to assess the DNA content of cells stained with PI, regions labeled M1, M2, M3, and M4 represent cells in Sub-G₁, G₁, S, and G₂/M phases of the cell cycle, respectively. (C) Cell cycle analyses were performed as in panel (B), with treatments for 24 (white bars), 48 (gray bars), and 72 h (black bars) and the percentage of cells in the G₁ phase of the cell cycle were plotted. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05. (D) The effect of amuvatinib on DNA synthesis was determined by treating U266 cells with DMSO or 5 and 25 μM amuvatinib for 24 (white bars), 48 (gray bars), and 72 h (black bars) and quantifying thymidine incorporation. The data are presented as percentages of time-matched controls. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05, **P < 0.01.