Fig 1: Bid neutralizes MuD function in TRAIL-mediated apoptotic signaling. (a, b) Bid siRNA1 (#681, sense strand: 5'-GGCAGAUUCUGAAAGUCAATT-3' anti-sense strand: 5'-UUGACUUUCAGAAUCUGCCTT-3') and Bid siRNA2 (#1085, sense strand: 5'-CGAUGUGGUCACAGCUGUATT-3' anti-sense strand: 5'-UACAGCUGUGACCACAUCGTT-3') target-specific 21-nt siRNAs designed to silence gene expression were obtained from BIONEER. Control and MuD siRNA were used as described in Figures 3d and f. pEGFPC1-MuD transfectants and T98G cells were seeded at a density of 3 × 105 cells in 6-well plates, respectively, and transfected with control, MuD, and Bid siRNA duplexes and MuD plus Bid siRNA using Lipofectamine2000. Following 36 h incubation, 2 × 104 cells were re-seeded into 96-wells, incubated for 12 h, and then treated with TRAIL (200 ng/ml) for an additional 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, **P<0.01). The rest of each cell were lysed, subjected to 12% SDS-PAGE, and the patterns of MuD expression were analyzed by western blot using C22B3 MAb (top panel) and anti-Bid Ab (middle panel). The blot was re-probed with anti-ß-actin (bottom panel). (c) U251-MG cells were seeded at a density of 3 × 105 cells in 6-well plates. The cells were transfected with control and Bid siRNA duplexes (#681), respectively, using Lipofectamine2000. Following 36 h incubation, the cells were lysed, subjected to 12% SDS-PAGE, and amount of MuD and Bid was analyzed by western blot using C22B3 MAb (top panel) and anti-Bid Ab (middle panel). The blot was re-probed with anti-ß-actin (bottom panel). (d) SV-40-transformed (control), Bid+/+ and Bid-/- mouse embryonic fibroblast (MEF) lysates were subjected to 12% SDS-PAGE and analyzed by western blot using C22B3 MAb (top panel) and anti-Bid Ab (middle panel). The blot was re-probed with anti-ß-actin (bottom panel). (e) pEGFPC1-MuD transfectants were seeded at a density of 3 × 105 cells in 6-well plates and transfected with control and MuD siRNA duplexes using Lipofectamine2000. Subsequent to 36 h incubation, cells were lysed and subjected to 12% SDS-PAGE. Amount of MuD and Bid was analyzed by western blot. (f) U251-MG cells were grown to 80% confluence in 100-mm culture dishes and then stimulated with TRAIL (200 ng/ml) for the indicated times (0–12 h). The cytoplasmic, mitochondrial and nuclear fractions were isolated from U251-MG cells using a cell fractionation kit (ab109719, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Fractions were subjected to 12% SDS-PAGE, and analyzed by western blot using C22B3 MAb to determine the subcellular localization of MuD. The blots were re-probed with anti-Cox IV, anti-tubulin (Abcam) and anti-lamin B (GeneTex, Irvine, CA, USA), respectively. (g) The microsomal fraction was isolated from the cultured cells after TRAIL treatment (200 ng/ml) for 0–12 h using an ER isolation kit according to the manufacturer's instructions (ER0100, Sigma, Saint Louis, MO, USA). Sample lysates were analyzed by 12% SDS-PAGE followed by western blot using C22B3 MAb for MuD detection. The blots were re-probed with anti-calnexin (Abcam), anti-Cox IV and anti-lamin B, respectively. (h) Proposed mechanism underlying the role of MuD in TRAIL-mediated death signaling. MuD resides at the ER and mitochondria regulates apoptotic signaling induced by the formation of TRAIL/TRAIL-R complex. Bid may be essential for the regulation of MuD expression and function. In addition, the formation of 25-kDa Bcl-2 fragments by TRAIL stimulation which converts Bcl-2 from its anti-apoptotic form to the truncated pro-apoptotic form is inhibited by MuD, affecting regulation of caspase activation which subsequently leads to apoptosis.