Effect of Notch-1 overexpression on p21 accumulation and drug resistance. Overexpression of Notch-1 results in accumulation of p21 and drug resistance of myeloma cells. (A) U266 cells were infected with retroviruses containing either pMSCV-IC-Notch-1-GFP construct or control vector pMSCV-IRES-GFP. At 24 hours later, cells were exposed to melphalan or vehicle control for an additional 24 hours. Apoptosis was measured by annexin V-binding assay within the population of GFP⁺ cells. The level of spontaneous apoptosis in cells treated with vehicle alone was subtracted. Two experiments with the same results were performed. (B) U266 cells were electroporated with pcDNA3.1-IC-Notch-1 or control pcDNA3.1 plasmid. At 24 hours later, dead cells were eliminated by gradient centrifugation. Apoptosis was measured in the total cell population by annexin V staining. Two experiments with the same results were performed. Error bars indicate standard deviation. (C) U266 cells were transfected with IC-Notch-1 as described for panel B, dead cells were removed, and cells were cultured for an additional 24 hours. After that time, cells were collected and lyzed, and p21 expression was analyzed by Western blotting.