Figure 5

Amuvatinib is tumoricidal to U266 myeloma cells. Apoptosis was quantified by staining cells with annexin V/PI and measuring staining positivity using flow cytometry. (A) A representative annexin V/PI staining profile 72 h after treatment with 25 μM amuvatinib or DMSO is shown. (B) U266 cells were treated with DMSO or various concentrations of amuvatinib for 24 (white bars), 48 (gray bars), and 72 h (black bars) and percent of annexin V/PI positivity is presented. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05. Immunoblot analysis demonstrating cleavage of PARP protein after treatment with amuvatinib at indicated concentrations under conditions of (C) full serum (10% FBS) for 24 h, or (D) serum starved conditions with endogenous HGF for 16 h, (E) U266 and RPMI-8226/S cells were treated with various concentrations of amuvatinib for 48 h and percent of annexin V/PI positivity is presented. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05. (F) U266 cells cultured alone (black bars), or on NK-tert stromal cells (gray bars), or stromal cells alone (white bars), were treated with or without 25 μM amuvatinib for 48 h and assessed by flow cytometry for annexin V/PI staining. (G) Cells were treated with various concentrations of imatinib and annexin V/PI positivity is presented as percentage of time-matched controls. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05.