GSI effect on MM cells is mediated through Hes-1. (A,B) H929 cells were treated with GSI for 4, 8, or 24 hours. After that time, cells were collected and (A) the expression of Hes-1 gene was determined by real-time PCR as described in “Quantitative real-time polymerase chain reaction” and normalized to the expression of housekeeping gene 18S. * indicates statistically significant difference (P < .05) in 2-tailed t test. (B) In parallel, cells were stained with annexin V/7-AAD and the level of apoptosis was detected by flow cytometry. (C) Overexpression of Hes-1 abrogated GSI-induced apoptosis in MM cells. Human U266 MM cells were transfected with either pIRES2-AcGFP-Hes-1 or control empty pIRES2-AcGFP vector and treated with the indicated concentrations of GSI for 24 hours. Apoptosis was measured by flow cytometry in a gated GFP-positive population of cells by annexin V-PE/7-AAD staining using FACSCalibur. Error bars represent SD.