1. Volume 14, Issue 5, Pages 585-598 (June 2004)
The mRNA Surveillance Protein hSMG-1 Functions in Genotoxic Stress Response Pathways in Mammalian Cells 
Kathryn M. Brumbaugh, Diane M. Otterness, Christoph Geisen, Vasco Oliveira, John Brognard, Xiaojie Li, Fabrice Lejeune, Randal S. Tibbetts, Lynne E. Maquat, Robert T. Abraham 
Molecular Cell 
Volume 14, Issue 5, Pages (June 2004)
DOI: /j.molcel

2. Figure 1 hSMG-1 Expression and Kinase Activity
(A) Expression of recombinant and endogenous hSMG-1. (Left panel) HEK293T cells were either left untransfected (−) or were transfected with empty vector, HA-SMG-1wt, or HA-SMG-1KI-encoding plasmid DNA. After 24 hr, the transfected cells were lysed, and soluble proteins were immunoprecipitated (IP) with preimmune serum, α-SMG-1 (Ab-2), or α-HA antibodies. (Right panel) Whole-cell extracts (WCE) were separated by SDS-PAGE. The membranes were immunoblotted (IB) with α-SMG-1 (Ab-1).
(B) hSMG-1 kinase activity. HEK 293T cells were transfected with the indicated expression plasmids, and α-HA immunoprecipitates were subjected to immune complex kinase assays with GST-p531–70, GST-p531–70 Ser-15→Ala (S15A), or GST-hUpf11019–1118 as substrate in the presence of [γ-32P]ATP. The reaction products were immunoblotted with α-HA to determine the levels of recombinant hSMG-1 or ATM in each sample (lower panel).
(C) Radiation-induced hSMG-1 activation. HEK 293T cells were transfected with either empty vector or HA-SMG-1wt expression vector. After 48 hr, the cells were treated with 10 Gy IR or 100 J/m2 UV-B light. Cells were harvested and detergent extracts were prepared at 1 or 2 hr postirradiation. Kinase activities in α-HA immunoprecipitates were measured with GST-p531–70 as the substrate. Phosphorylation of the substrate was determined by immunoblotting with a pSer-15-specific antibody, quantitated by densitometric analysis with NIH image analysis (version 1.62) software, and normalized to the sample from nonirradiated cells. The right panel shows hSMG-1 activation results (mean ± SEM) from five independent trials.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Role of hSMG-1 in Stress-Induced p53 Phosphorylation and Accumulation
(A) hSMG-1 overexpression. U2OS cells were cotransfected with a GFP-NLS, together with empty vector, or with HA-SMG-1wtT- or HA-SMG-1KI-encoding plasmid. After 40 hr, the cells were exposed to 10 Gy IR, and GFP-positive cells were collected after 4 hr with a fluorescence-activated cell sorter. Whole-cell extracts were resolved by SDS-PAGE and sequentially immunoblotted with the indicated antibodies.
(B) hSMG-1 depletion by siRNA. U2OS cells were mock transfected or transfected with hSMG-1 siRNA. After 48 hr, the cells were treated with 10 Gy IR or 200 J/m2 UV and were lysed after 4 hr. Soluble proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies.
(C) ATM versus hSMG-1 depletion. U2OS cells were transfected with hSMG-1 or ATM siRNA, either singly or in combination. After 72 hr, the cells were treated with 3 or 10 Gy IR and harvested at 1 hr or 4 hr postirradiation. Detergent-soluble proteins were separated by SDS-PAGE and were immunoblotted with the indicated antibodies. LEVEL denotes p53 protein expression normalized to the nonirradiated, luciferase siRNA-treated control sample.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Histone H2AX Phosphorylation
(A) γH2AX-positive nuclear foci. U2OS cells were treated with the indicated siRNA and were stained with γH2AX-specific antibody (red). Cell nuclei were counterstained with DAPI (blue). The bottom panel shows percentages of γH2AX-positive nuclei (mean ± standard deviation) in each cell population.
(B) γH2AX immunoblotting. U2OS cells were transfected with luciferase or hSMG-1 siRNA and were exposed to the indicated doses of IR. The cells were harvested after 1 hr or 18 hr, and detergent-soluble proteins were separated by SDS-PAGE through a 4%–20% gradient gel (Novex). Protein blots were sequentially probed with γH2AX antibody followed by α-tubulin antibodies to control for sample loading in each lane.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Effect of hSMG-1 Depletion on Cell Cycle Progression
(A) Cell cycle distribution. U2OS cells were either nontransfected or transfected with sense (S) or antisense (AS) oligonucleotide targeted against hSMG-1. After 40 hr, cells were exposed to 20 Gy IR. Cell cycle distributions were examined after 16 hr by flow cytometry. The table shows the percentage of cells in each cell cycle phase. The right panel shows immunoblot analyses from the same cell populations.
(B) Effect of caffeine. U2OS cells were transfected with S or AS oligonucleotide as described above. The cells were treated for 8 hr with 2 mM caffeine and were then harvested for cell cycle distribution analyses. The table shows percentages of cells in each cell cycle phase, plus the ratio of G2/M to G1 cells for each sample. The right panel depicts immunoblotting results from the same cell populations.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Radiation Sensitivity of hSMG-1-Depleted Cells
A549 cells were transfected with luciferase or hSMG-1 siRNA and replated after 24 hr for clonogenic survival assays. After 48 hr, the cells were exposed to the indicated doses of IR, and surviving colonies counted after 12 days in culture. The immunoblot shows hSMG-1 levels in the test cell populations at 72 hr posttransfection.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 IR-Induced hUpf1 Phosphorylation
(A) Effect of IR or OA. U2OS cells were transfected with a FLAG-hUpf1, and, after 24 hr, cells were serum starved for 16 hr. Cells were then treated with 250 nM OA and/or 10 Gy IR, and cellular extracts were immunoprecipitated with α-FLAG-agarose beads. Immunoprecipitated proteins were separated by SDS-PAGE and sequentially probed with α-pS/T-Q antibody, followed by α-FLAG antibody. Immunoblots were analyzed by densitometry, and α-pS/T-Q immunoreactivity was normalized to the level of α-FLAG immunoreactivity for the same sample. The signal ratio was obtained by normalizing these values to the nonirradiated control.
(B) Effect of hSMG-1 versus ATM depletion. U2OS cells were transfected with the indicated siRNA, and after 72 hr, the cells were exposed to IR or OA. The cells were lysed after 1 or 4 hr, and immunoblotting was done with the indicated antibodies. Signal ratios were calculated as described in (A).
(C) Phosphorylation of endogenous hUpf1. U2OS cells were treated for the indicated times with IR or OA, and α-hUpf1 immunoprecipitates were sequentially immunoblotted with α-pS/T-Q and α-hUpf1 antibodies (left panel). Cell extracts were also immunoprecipitated with α-S/T-Q antibodies and immunoblotted with either α-hUpf1 or α-NBS1 antibodies (right panel).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
NMD Assays
(A) Effect of hSMG-1 depletion. U2OS cells were cotransfected with Norm- or Ter-containing β-globin test plasmid, a MUP reference plasmid, and either sense (S) or antisense (AS) oligonucleotide directed against hSMG-1. At 48 hr posttransfection, cells were harvested, nuclear RNA was isolated, and β-globin and MUP mRNAs were quantitated by RT-PCR and phosphorimaging. For each pair of transfections, the level of globin Ter mRNA was normalized to the level of MUP mRNA and expressed below each lane as a percentage of the normalized level of globin Norm mRNA, which was defined as 100. Results shown are representative of two independent experiments.
(B) Effect of hSMG-1 versus ATM depletion. Calu6 cells were transfected with hSMG-1 or ATM siRNA and total cellular RNA was isolated after 48 hr. The indicated sample was treated for 6 hr with 5 μM wortmannin prior to harvest. Levels of PTC-containing p53 (1–195) mRNA transcripts were determined in triplicate samples by quantitative (Q)-PCR. Histogram depicts fold increase (mean ± SEM, n = 3 samples), normalized to the luciferase siRNA-transfected samples. The left panel shows immunoblot results from parallel samples.
(C) NMD in IR-treated cells. Calu6 cells were transfected with the indicated siRNAs, and after 72 hr, were exposed to 0 or 10 Gy IR. The cells were harvested after 6 hr, and Q-PCR and immunoblot analyses were performed as described in (B).
Molecular Cell  , DOI: ( /j.molcel )