Treatment with GSI induces MM cell death. (A,B) H929, U266, MM1S, or RPMI 8226 cells (5 × 10⁴), 10⁵ myeloma cells obtained from BM aspirates of 4 MM patients, or 10⁵ BM cells from 4 healthy donors or PB MNCs obtained from 5 donors were plated per well of 96-well plates and treated with GSI for 48 hours followed by the MTT test. (A) Cell viability curves for MM cell lines and normal BM cells are shown. (B) IC₅₀ values were calculated for all types of cells studied. The differences between the values in MM cell lines and primary MM cells and control BM or PB MNCs were statistically significant in 2-tailed t test (P < .002). (C) H929 MM cell line or (E) primary CD138⁺ myeloma cells were treated with GSI or vehicle control for 24 hours. Proportion of apoptotic cells was analyzed by annexin V-PE/7-AAD staining using FACSCalibur flow cytometer. (D) The expression of Notch ligands on MM cells was analyzed by Western blotting. Membranes were reprobed with β-actin antibody to confirm protein loading. (F) CD34⁺ cells were isolated from the BM of healthy donors and cultured on the monolayer of BMS for 24 hours. After that time, CD34⁺ cells were collected and plated in semi-solid media according to the manufacturer's protocol. A total of 5000 CD34⁺ cells were plated per well and cultured either in the presence of DMSO (control) or 8 μM GSI. Experiments were performed in duplicate and repeated once. Colonies were scored using an inverted microscope. The numbers of colonies per 5 × 10³ CD34⁺ cells are shown. CFU indicates colony-forming units; GM, granulocyte/macrophage; M, macrophage; E, erythrocyte; GEMM, mixed; and BFU-E, burst forming units erythroid. Error bars represent SD.