Treatment with GSI blocks Notch pathway activation in MM cells. (A) H929 cells stably transfected with the CBF-1 reporter or control vector were cultured on monolayer of BMS for 48 hours in the presence of vehicle control (DMSO, vc) or GSI. After that time, MM cells were collected and lysed using the reporter lysis buffer (Promega). The luciferase activity (LA) was normalized to 1 μg of total protein. Data are presented as fold increase of the specific LA (H929 cells transfected with p6 × CBF1-pKA9 plasmid) over control LA activity (cells transfected with pKA9 plasmid). Experiments were repeated on BMS from 3 different donors with similar results. (B,C) Myeloma H929 and 8226 cell lines (B) or primary CD138⁺ MM cells isolated from the BM of patients with MM (C) were cultured either in the presence of DMSO (vehicle control, vc) or GSI for 4 hours and 16 hours, respectively. The level of Hes-1 expression was determined in triplicate by real-time PCR and was normalized to the expression of housekeeping gene 18S (*P < .05, statistically significant difference in 2-tailed t test). Error bars represent SD.