Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 2. Knockdown of PRMT5 potentiates TRAIL-induced apoptosis in cancer cells. A. HeLa cells were transfected with control, PRMT5-1, or PRMT5-3 siRNA. Cell lysates were immunoblotted with the indicated antibodies. B. HeLa cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL in the presence or absence of 10 μmol/L Z-VAD for 24 h. Cell viability was evaluated using the MTS assay in a percentage of the control siRNA-transfected cells without TRAIL treatment. *, P = 0.0016; **, P < 0.00005, compared with TRAIL-untreated and the same siRNA-transfected cells; #, P > 0.05, compared with TRAIL-untreated and the same siRNA- and Z-VAD-treated cells. C. HeLa, A549, HCT116, and HT1080/DR4 cells were transfected with the indicated siRNA and then treated with the indicated concentrations of TRAIL. Cell viability was evaluated using the MTS assay in a percentage of the corresponding cells without TRAIL treatment. *, P < 0.05; **, P < 0.01, compared with control siRNA-transfected and the same concen- tration of TRAIL-treated cells. D. HeLa cells were transfected with control (left) or PRMT5-3 (right) siRNA and then treated with a vehicle (open areas) or 100 ng/mL TRAIL (closed areas) for 24 h. Cells were stained with Annexin V-phycoerythrin, and the fluorescence was analyzed using a flow cytometer. E. WI- 38 and TIG3 fibroblast cells were transfected with the indicated siRNA. Cell lysates were immunoblotted with the indicated antibodies. F. WI-38 (left) and TIG3 (right) cells were transfected with the indicated siRNA and then treated with the indicated concentrations of TRAIL. Cell viability was evaluated using MTS assay. #, P > 0.05, compared with TRAIL-untreated and PRMT5-3 siRNA-transfected cells. Bars, SD of triplicate experiments (B, C, and F).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Knockdown, Transfection, Control, MTS Assay, Staining, Fluorescence, Flow Cytometry

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 3. PRMT5 confers TRAIL resistance independent of its methyltransferase activity. A, D, and F. HCT116 clones stably transfected with pcDNA3- Myc (Mock1, Mock3, and Mock4), pcDNA3-Myc-WT-PRMT5 (WT2, WT24, and WT42), pcDNA3-Myc-MD-PRMT5 (MD6, MD28, and MD32), or pcDNA3-Myc- ΔC325-PRMT5 (ΔC9, ΔC16, and ΔC25) were treated with the indicated concentrations of TRAIL (A) or 30 ng/mL TRAIL (D and F). Cell viability was evaluated using the MTS assay in a percentage of the corresponding cells without TRAIL treatment. Bars, SD of multiple experiments [A and F (n = 3) and D (n = 5)]. B. HeLa cells were transiently transfected with pcDNA3-Myc vectors encoding none (-), WT-PRMT5 (WT), or MD-PRMT5 (MD). Myc-tagged proteins were immunoprecipitated and subjected to an in vitro methyltransferase assay using recombinant H2A (rH2A) as a substrate (top). Immunopreci- pitated proteins were immunoblotted with an antibody to Myc tag (bottom). C, E, and G. Cell lysates of the stable transfectants were immunoblotted with the indicated antibodies.

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activity Assay, Clone Assay, Stable Transfection, Transfection, MTS Assay, Immunoprecipitation, In Vitro, Recombinant

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 4. Involvement of PRMT5 in TRAIL-induced IKK activation and IκB degradation. A. HeLa cells were transfected with the indicated siRNA and then incubated with control mouse IgG or antibodies to DR4 (left) or DR5 (right). Fluorescence was analyzed using a flow cytometer. Broken lines, fluores- cence of control IgG-treated and control siRNA-treated cells; open and closed areas, control and PRMT5-3 siRNA-treated cells, respectively. B. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 30 min. V5-tagged DR4 proteins were immunoprecipitated with anti-V5 agarose. Immunoprecipitated proteins (IP) and cell lysates (Input) were immunoblotted with the indicated antibodies. C. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL or 20 ng/mL TNF-α for the indicated times. Cell lysates were immunoblotted with the indicated antibodies (left). Relative intensities of the phospho-IκBα bands normalized with total IκBα bands were quantified (right). D. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 30 min. Endogenous IKK complexes were immunoprecipitated with anti-NEMO agarose and incubated with GST-tagged recombinant IκBα (rIκBα) as described in Materials and Methods. Control experiments were done with control rabbit IgG agarose. Reactions (in vitro kinase assay) and cell lysates were immunoblotted with the indicated antibodies. Relative intensities of the phospho-IκBα bands were quantified. E. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 30 min. Cell lysates were immunoblotted with the indicated antibodies.

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activation Assay, Transfection, Incubation, Control, Fluorescence, Flow Cytometry, Immunoprecipitation, Recombinant, In Vitro, Kinase Assay

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 5. TRAIL-induced NF-κB activation is dependent on PRMT5. HT1080/DR4 (A, left, and B) or HCT116 (A, right) cells were trans- fected with the indicated siRNA. After transfection for 24 h, cells were then transfected with pNF-κB-Luc and phRL-TK plasmids. After a further 24 h incubation, cells were treated with 100 ng/mL TRAIL (A) or 20 ng/mL TNF-α (B) for 4 h. Luciferase activities were measured as described in Materi- als and Methods. C. HT1080/DR4 cells were transfected with pNF-κB-Luc and phRL-TK plasmids. Four hours later, cells were treated with the 50 μmol/L methyltransferase inhibitor periodate- oxidized adenosine (AdOx) for 44 h. Then, cells were treated with 100 ng/ mL TRAIL for 4 h. Luciferase activities were measured. Bars, SD of triplicate experiments (A-C).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activation Assay, Transfection, Incubation, Luciferase

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 6. PRMT5-mediated transcription of NF-κB target genes. A to C. HCT116 (left) or HT1080/DR4 (right) cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 3 h. Total RNA was separa- ted and analyzed using quantitative RT-PCR (A and B) as described in Materials and Methods. Levels of cIAP1 (A) and cIAP2 (B) mRNA were normalized to the level of GAPDH mRNA. *, P < 0.005; **, P < 0.0005, compared with control siRNA-transfected and TRAIL-treated cells. Bars, SD of triplicate experiments (A and B). Cell lysates were immunoblotted with the indicated antibodies (C).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Transfection, Quantitative RT-PCR, Control

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 7. PRMT5 contributes to TRAIL resistance via NF-κB activation. A. HCT116 (left) or HT1080/DR4 (right) cells were treated with 10 ng/mL TRAIL or 20 ng/mL TNF-α in the presence or absence of the 10 μmol/L IKK inhibitor BMS-345541. Cell viability was evaluated using the MTS assay. **, P < 0.001, compared with TRAIL-untreated or TNF-untreated and BMS-345541-treated cells. B. HCT116 (left) or A549 (right) cells were transfected with the indicated siRNA and then treated with 10 ng/mL TRAIL or 100 ng/mL TNF-α. Cell viability was evaluated using the MTS assay. *, P < 0.05; **, P < 0.001; #, P > 0.05, compared with TRAIL or TNF-untreated and PRMT5-3 siRNA-transfected cells. C. HT1080 cells were transfected with control (-) or PRMT5-3 (+) siRNA. After transfection for 24 h, cells were also transfected with pEGFP-C1 (-) or pEGFP-C1-CA-IKKβ (+). After additional incubation for 24 h, cells were treated with 100 ng/mL TRAIL for 6 h. Cells were fixed and stained with Hoechst 33342. Cellular images were visualized using a fluorescence microscope. The rate that apoptotic cells showed condensed and fragmented nuclei was determined by counting >100 GFP-positive cells (left). Green, GFP proteins; blue, nuclei (right). *, P = 0.037; **, P = 0.00061, compared with control siRNA-transfected and TRAIL-treated cells, respectively; †, P = 0.0054, compared with IKKβ- untransfected, PRMT5-3 siRNA-transfected and TRAIL-treated cells. Bars, SD of triplicate experiments (A-C).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activation Assay, MTS Assay, Transfection, Control, Incubation, Staining, Fluorescence, Microscopy