Loss of hSMG-1 selectively sensitizes U2OS cells to TNFα-induced apoptosis. A, selective induction of apoptosis by hSMG-1 gene silencing. U2OS cells were transfected with the indicated siRNAs, and, after 48 h, the cells were stimulated for 2 h with 20 ng/ml TNFα. Percentage of cell death was determined by trypan blue dye exclusion. Results (mean ± S.E.) were obtained from four independent trials. Whole cell extracts from parallel samples were resolved by SDS-PAGE and immunoblotted with α-caspase-8 antibody. B, reversal of TNFα-induced cell killing in hSMG-1-depleted cells by forced expression of hSMG-1. U2OS cells were transfected with luciferase (Luc)- or hSMG-1-targeted siRNA, followed by secondary transfection with the indicated amounts of expression plasmids encoding HA-tagged, wild-type hSMG-1 (HA-hSMG-1^WT) or kinase inactive hSMG-1 (HA-hSMG-1^KI). HA-hSMG-1^KI contains an Asp → Ala substitution at a conserved residue (Asp-2195) in the hSMG-1 catalytic domain. The cells were stimulated with TNFα at 48 h after the initial transfection, and cell death was scored as described in A. Results are depicted as mean ± S.E. from three independent trials. The bottom panel shows immunoblots of the endogenous and ectopically expressed (HA-tagged) hSMG-1 proteins.

Rate of protein translation in hSMG-1-depleted cells. U2OS cells were treated with luciferase- or hSMG-1-siRNAs for 72 h, starved in fetal bovine serum-, methionine-, and cysteine-free medium for 3 h, labeled with [³⁵S]methionine/cysteine, and harvested at the indicated times. Cycloheximide (CHX)-treated cells were subjected to a similar experimental protocol, and trichloroacetic acid-precipitable radioactivity was measured by scintillation counting. Results (mean ± S.E.) were obtained from three independent trials.

DISC composition in hSMG-1-depleted cells. A, DISC I. U2OS cells were treated with luciferase or hSMG-1 siRNAs for 48 h, stimulated with recombinant FLAG-tagged human TNFα (FLAG-TNFα), and immunoprecipitated with a FLAG-specific monoclonal antibody. Samples were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. B, DISC II. After FLAG-TNFα stimulation, extracts from luciferase and hSMG-1-depleted cells were immunoprecipitated with α-caspase-8 antibody and immunoblotted with the indicated antibodies. The arrows point to proteolytically cleaved proteins. C, cellular extracts are shown that correspond to 1/100th of the total lysate used in A and B.

hSMG-1 depletion sensitizes U2OS cells to TNFα-induced killing. A, TNFα-induced cell death. Cells were transfected with luciferase or hSMG-1 siRNA, and, after 48 h, were exposed to 20 ng/ml TNFα for the indicated times. Cell viability was determined by trypan blue dye exclusion. Results (mean ± S.E.) were obtained from three independent trials. B, hSMG-1 immunostaining. U2OS cells were treated with the indicated siRNAs for 48 h, exposed to TNFα for the indicated times, and stained with a polyclonal α-SMG-1 antibody (green). Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (blue), and images were obtained by immunofluorescence microscopy. C, cleaved caspase-3 levels. U2OS cells were treated with luciferase- or hSMG-1-siRNAs for 48 h, or left untreated, and were exposed to 20 ng/ml TNFα and/or 10 μg/ml cycloheximide (CHX). At the indicated times, the relative amount of cleaved caspase-3 was determined with a cleaved caspase-3 assay (meso scale discovery). Data are presented as mean ± S.D. from triplicate samples. D, DNA fragmentation. U2OS cells were transfected with the indicated siRNAs, and, after 48 h, the cells were stimulated for 2 h with 20 ng/ml TNFα and/or 10 μg/ml CHX. The cells were then harvested, and DNA fragmentation was evaluated with the Cell Death Detection ELISA plus kit (Roche Applied Science). Data are presented as mean ± S.D. from triplicate samples. E, caspase-8 cleavage. U2OS cells were treated with 10 μg/ml CHX and/or 20 ng/ml TNFα and were harvested at the indicated times. Detergent-soluble proteins were resolved by SDS-PAGE and immunoblotted with α-hSMG-1, α-PLC-γ1, and α-caspase-8 antibodies. In the α-caspase-8 immunoblot, the p43/41 bands denote the cleaved forms of caspase-8. F, the experiment was performed as described in C, except that the cells were transfected with the indicated siRNA prior to TNFα stimulation. G, BAX activation. Whole cell extracts were prepared from siRNA-transfected U2OS cells, and detergent extracts were immunoprecipitated with an antibody that selectively recognizes the active conformation of BAX (α-Active BAX).

Effect of hSMG-1 versus hUpf-1 depletion on the PTC-dependent degradation of β-globin mRNA. A, β-globin NMD reporter. A schematic diagram of the structure of human β-globin-luciferase reporter constructs, Luc-BGG-WT and Luc-BGG-39PTC, is shown. B, NMD reporter assays. U2OS cells were stably transfected with wild-type or PTC-containing β-globin reporter plasmid and a TK-Renilla reference plasmid. Stable clones were then treated with the indicated siRNAs, or 10 μm Wortmannin (Wm) for 12 h. DMSO was added to the indicated sample as a vehicle control (VC). At 72 h post-transfection, cells were harvested, and luciferase activities were measured with a luciferase assay system (Promega), and each sample was normalized to the Renilla luciferase control activity.

NF-κB pathway activation in hSMG-1-depleted cells. A, IκBα degradation. U2OS cells were treated with luciferase- or hSMG-1-siRNA oligonucleotides for 48 h, stimulated with TNFα, and detergent extracts were immunoblotted with the indicated antibodies. B, NF-κB reporter gene assay. U2OS cells were co-transfected with NF-κB-Luciferase reporter plasmid (NF-κB-Luc), a TK-Renilla reference plasmid, and increasing amounts of HA-tagged, wild-type TRAF2 (HA-TRAF2). At 48 h post-transfection, luciferase activities were measured and normalized as described in the Fig. 3 legend. Results (mean ± S.E.) were obtained from three independent trials. The immunoblot shows hSMG-1 and HA-TRAF2 levels in the test cell populations at 48 h post-transfection. C, NF-κB DNA binding activity. U2OS cells were treated as described in A, and nuclear extracts were analyzed for NF-κB DNA binding activity by electrophoretic mobility shift assay using a canonical κB site as a probe. Binding to a NF-1 probe was used as a loading control. The bottom panel shows expression levels of hSMG-1, total ERK, and phosphorylated ERK (P-ERK) at 48 h post-transfection. The arrows point to p44 and p42 MAPKs (i.e. Erk1 and Erk2, respectively).

TNFα-induced down-regulation of FLIP_L in hSMG-1 siRNA-treated cells. A, FLIP_L protein expression. U2OS cells were transfected with luciferase or hSMG-1 siRNAs for 48 h, and cells were exposed to 20 ng/ml TNFα. After 90 min, the cells were harvested, and lysed by Dounce homogenization in hypotonic buffer. Subcellular fractions were prepared as described under “Experimental Procedures.” Protein levels of Topo IIβ, Bcl-2, and PLC-γ1 were determined to show equivalent loading of nuclear, membrane, and cytosolic fractions, respectively. B, effect of proteasome inhibitors on FLIP_L expression. Cells were transfected and treated with TNFα as described in A. The indicated samples were exposed for 8 h to 5 ng/ml PS-341 or for 4 h to 15 μm MG132 prior to TNFα stimulation. Detergent-soluble membrane proteins were immunoblotted as described in A. C, effect of hSMG-1 depletion on TNFα-mediated FLIP_L degradation. U2OS cells were transfected with luciferase or hSMG-1 siRNAs for 48 h. Transfected cells were treated for the indicated times with 100 μg/ml CHX (upper panels), or were pretreated for 30 min with CHX, then stimulated with 20 ng/ml TNFα for the indicated times (lower panels). Detergent-soluble proteins were resolved by SDS-PAGE and immunoblotted with α-hSMG-1, α-FLIP_L, and α-β-actin antibodies. D, FLIP_L mRNA expression. U2OS cells were treated with luciferase- or hSMG-1-siRNA duplexes, as described in A. After TNFα stimulation, levels of FLIP_L mRNA transcripts were determined in triplicate samples by real-time quantitative PCR. Results (mean ± S.E.) were normalized to glyceraldehyde-3-phosphate dehydrogenase expression. The right panel shows immunoblots of hSMG-1 and β-actin proteins at 48 h post-transfection. E, expression of recombinant FLIP_L. U2OS cells were treated with the indicated siRNA, and after 24 h, the cells were transfected with empty vector or FLAG-tagged, wild-type FLIP_L (Flag-FLIP_L). At 72 h post-siRNA transfection, the cells were stimulated for 2 h with 20 ng/ml TNFα, and the percentage of cell death was determined by trypan blue dye exclusion. Results (mean ± S.E.) were obtained from three independent trials. In parallel, cell extracts were prepared and immunoblotted with the indicated antibodies.