Figure 3.

MuD exerts an inhibitory effect on TRAIL-induced sensitivity to death stimuli. (a) U251-MG cells stably expressing GFP alone (pEGFPC1) and GFP-MuD (pEGFPC1-MuD) were generated by transfection using Lipofectamine2000 and selected on G418 sulfate (200 μg/ml; Invitrogen). Amount of MuD was analyzed by western blot using C22B3 MAb and anti-GFP Ab (Santa Cruz). The blot was re-probed with anti-β-actin. (b) pEGFPC1 (white) and pEGFPC1-MuD (black) stable transfectants were stimulated by TRAIL for the indicated time periods (0–12 h). Cell viability was measured using WST-1. Data presented are the mean±s.d. of three experiments (significant versus control, *P<0.05). (c) pEGFPC1 and pEGFPC1-MuD transfectants were seeded at a density of 3 × 10⁶ cells in 100-mm culture dishes and incubated overnight. Cells were then treated with TRAIL (200 ng/ml) for the indicated times (0–180 min). Cell lysates were separated on 10% SDS-PAGE and analyzed by western blot with the following antibodies; C22B3 MAb, anti-Bid, anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-caspase-9, anti-Bcl-2 (Santa Cruz) and β-actin as a loading control. (d, f) MuD siRNA (sense strand: 5′-GAGCAAGUUAUGUGCCUGUdTdT-3′ anti-sense strand: 5′-ACAGGCACAUAACUUGCUC-3′), a target-specific 19-nt siRNA designed to silence the gene expression and control siRNA (sense strand: 5′-CCUACGCCACCAAUUUCGU-3′ anti-sense strand: 5′-ACGAAAUUG GUGGCGUAGG-3′) were purchased from BIONEER. U251-MG (d) and T98G cells (f) were seeded at a density of 3 × 10⁵ cells in 6-well plates, respectively. Twelve hours after seeding, cells were transfected with control and MuD siRNA duplexes (100 nM) using Lipofectamine2000. Following 36 h incubation, 2 × 10⁴ cells were then re-seeded into 96-wells, incubated for 12 h, and then treated with TRAIL (200 ng/ml) for the indicated time periods (0–12 h). Cell viability was measured using WST-1. Data presented are the mean±s.d. of three experiments (significant versus control, *P<0.05). (e, g) Each cell from the experiment presented in d and f were lysed, subjected to 10% SDS-PAGE, and amount of MuD was analyzed by western blot using C22B3 MAb. The blot was re-probed with anti-β-actin. (h) U251-MG cells were seeded at a density of 3 × 10⁶ cells in 100-mm cell culture dishes. Following overnight incubation, the cells were transfected with control and MuD siRNA duplexes (100 nM) using Lipofectamine2000, respectively. Subsequent to 36 h incubation, the cells were re-seeded into 6 wells (3 × 10⁵ cells/well), incubated for an additional 24 h, and then stimulated with TRAIL for the indicated times (0–180 min). The expression patterns of MuD and the caspases were detected by western blot analysis with the following antibodies; C22B3 MAb, anti-Bid, anti-caspase-9, anti-caspase-3, anti-Bcl-2 and β-actin as a loading control. FL, full length.