Effect of Notch-1 pathway activation on p21 and cell growth. Activation of Notch-1 pathway results in up-regulation of p21 and growth arrest in myeloma cells. H929, RPMI 8226, U266, or HS-Sultan myeloma cells were incubated on the monolayer of irradiated 3T3-Jagged or 3T3-MSCV fibroblasts (A,C-D), or BMS cells (B), or kept in suspension for 48 hours. (A-B) [³H]-thymidine was added for the last 16 hours of culture. Thymidine incorporation was measured by liquid scintillation counting. Background level of thymidine incorporation into irradiated stromal cells was subtracted in all experiments. (C-D) Myeloma cells were collected, fixed, and stained with propidium iodide (PI). Cell cycle distribution was analyzed by flow cytometry. (E) H929 cells were incubated with 3T3-Jagged (J) or 3T3-MSCV (Con) cells for 24, 36, or 48 hours. Myeloma cells were collected, and p21 and p27 protein expression was analyzed by Western blotting. To confirm equal loading, membrane was reprobed with β-actin antibody. (F-G) U266 cells were cultured on 3T3-Jagged (J) or 3T3-MSCV (Con) stroma (F); H929 cells were incubated on BMS or kept in suspension for 48 hours (G). Myeloma cells were collected, and p21 expression was analyzed by Western blotting. Error bars (A-D) indicate standard deviation.