Journal: bioRxiv

Article Title: PRMT5 as an Epigenetic Target for Group 3 (MYC-driven) Medulloblastoma

doi: 10.64898/2026.04.09.717536

Figure Lengend Snippet: (A) qPCR analysis for the expression of PRMT5/MYC mRNA in two MYC-amplified cell lines with transiently knocked-down of PRMT5 (using siRNAs) at 72 h. ****, p<0.0001 (student t test, SCR vs siPRMT5). (B) Effect of PRMT5 knockdown (siRNAs) on MYC-luciferase reporter gene (MYC-Luc) activity in HD-MB03 cells. *, p<0.05 (student t test) (C) ChIP analyses for the enrichment of PRMT5 and H4R3me2s on the proximal promoter region of the MYC gene in HD-MB03 cells. ****, p<0.0001 (student t test). ChIP analyses for the enrichment/binding of PRMT5 (D) and H4R3me2s (E) to the proximal promoter region of MYC gene, in PRMT5 knockdown (siRNAs) HD-MB03 cells. ( F ) Co-immunoprecipitation of BRD4 with PRMT5. ( G ) ChIP analyses for the enrichment/binding of PRMT5 and BRD4 to the proximal promoter region of MYC gene, in BRD4 knockdown (siRNAs) HD-MB03 cells. *, p<0.05; **, p<0.05 (student t test).

Article Snippet: Four PRMT5 inhibitors (EPZ015666, GSK332595, LLY-283, JNJ64619178) were purchased from MedChemExpress LLC or Selleckchem Company.

Techniques: Expressing, Amplification, Knockdown, Luciferase, Activity Assay, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: PRMT5 as an Epigenetic Target for Group 3 (MYC-driven) Medulloblastoma

doi: 10.64898/2026.04.09.717536

Figure Lengend Snippet: RNA-sequencing was used to assess global gene expression changes in HD-MB03 cells 24 h after treatment with DMSO (vehicle) or JNJ64619178 (1 µM). (A) Volcano plot displaying genes significantly upregulated or downregulated in response to PRMT5 inhibition. (B) Gene Ontology Biological Process (GO-BP) functional analysis for top 10 pathways altered by JNJ. (C) Gene sets enrichment analysis (GSEA) (with p<0.01 and FDR<0.2) for top 10 pathways/gene sets (including MYC-associated target gene sets, RNA splicing, metabolism) altered by PRMT5 inhibition. (D) Number of the aberrant splicing events in protein coding genes disrupted by PRMT5 inhibition. (E) GO-BP functional analysis for the aberrant splicing events altered by PRMT5 inhibition. (F) Volcano plot represents all the significant (p<0.05) splicing events. Selected genes from the indicated GO-BP functional classification are highlighted. Metabolism associated genes GGT6, GPT2 and C1QBP1 are identified.

Article Snippet: Four PRMT5 inhibitors (EPZ015666, GSK332595, LLY-283, JNJ64619178) were purchased from MedChemExpress LLC or Selleckchem Company.

Techniques: RNA Sequencing, Gene Expression, Inhibition, Functional Assay

Journal: bioRxiv

Article Title: PRMT5 as an Epigenetic Target for Group 3 (MYC-driven) Medulloblastoma

doi: 10.64898/2026.04.09.717536

Figure Lengend Snippet: (A) MTT assay showing the effects of PRMT5 inhibitors (1-50 µM) on cell growth of MYC-amplified MB (HD-MB03, D341), non-MYC MB (ONS-76) and normal human astrocyte (NHA) cell lines. (B) IC 50 values of PRMT5 inhibitors in the indicated cell lines. (C) Annexin-V assay showing effects of PRMT5 inhibitors (JNJ, EPZ) on apoptosis in HD-MB03 cells. **, p<0.01, ***, p<0.001 (relative to ‘0’ (vehicle control)). (D) Cell cycle profile in HD-MB03 cells treated with PRMT5 inhibitors (EPZ, JNJ). (E) Western blot analysis for the expression of the indicated key proteins in JNJ-treated HD-MB03 cells. ( F) Quantification of spheres following treatment of JNJ and EPZ in two PDX-derived MB cell lines (MED-411FH, MED-114FH) at 72 h. Values, mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; *** p < 0.001 (Student- t -test). (G) Representative sphere (MED-411FH) images showing disruption of spheres in each treatment. (H) Western blot results showing the expression of indicated proteins in MED-411 spheres treated with JNJ and EPZ.

Article Snippet: Four PRMT5 inhibitors (EPZ015666, GSK332595, LLY-283, JNJ64619178) were purchased from MedChemExpress LLC or Selleckchem Company.

Techniques: MTT Assay, Amplification, Annexin V Assay, Control, Western Blot, Expressing, Derivative Assay, Disruption

Journal: bioRxiv

Article Title: PRMT5 as an Epigenetic Target for Group 3 (MYC-driven) Medulloblastoma

doi: 10.64898/2026.04.09.717536

Figure Lengend Snippet: (A ) JNJ brain concentration in BALB/c mice (N=3) following oral administration of 10 mg/kg JNJ at different timepoints. **p<0.01 (Student’s t-test). (B) NSG mice (N=5) with subcutaneously xenografted HD-MB03 cells were treated orally with vehicle or JNJ (10 mg/kg) five times a week for three weeks. Tumor volume measurement of xenografted mice following treatments. The differences noted between treatment groups show comparison by Student ‘s t-test of the tumor volumes on 21 days post treatment (p<0.005). (C) Representative IHC images (40 × magnification with 60 µm scale bar) of PRMT5, MYC, Ki-67, and CC3 expression in xenografts 21 days post-treatment, as indicated. Bar graphs below show the percentages of PRMT5, MYC, Ki-67 and CC3 positive cells derived from immunohistology scores, which were semi-quantitated in the tumors of three xenografted mice. **p<0.01; ***p<0.005 (Student’s t-test). (D) NSG mice (N=6) with orthotopically xenografted HD-MB03 cells were treated daily with vehicle or JNJ (10 mg/kg) or JNJ (F) (10 mg/kg) for two weeks. Survival analysis of xenografted mice using Kaplan-Meier (long-rank test). *p<0.05, **p<0.01, ***p<0.001. (E) Representative IHC images (4x magnification with 600 µm scale bar) and respective quantification showing MYC-positive tumors in the mouse cerebellum. The percentage of MYC, derived from immunohistology scores, was semi-quantitated in the tumors of three xenografted mice 21 days post-treatment. *p< **p<0.01 (Student’s t-test).

Article Snippet: Four PRMT5 inhibitors (EPZ015666, GSK332595, LLY-283, JNJ64619178) were purchased from MedChemExpress LLC or Selleckchem Company.

Techniques: Concentration Assay, Comparison, Expressing, Derivative Assay

Journal: PLOS Biology

Article Title: CUL5 E3 ubiquitin ligase regulates the evasion of bladder cancer cells to CD8 + T cell-mediated killing by inhibiting autophagy

doi: 10.1371/journal.pbio.3003647

Figure Lengend Snippet: (A) Silver staining showed the proteins pulled down by CUL5 from the lysates of T24 cells. (B) Analysis pipeline was performed to identify potential proteins that interact with CUL5: (i) The proteins with unique peptides ≥5 and fold change ≥20 in T24-MS were selected; (ii) The RNA splicing biological process exhibited the most significant enrichment within the protein-protein interaction (PPI) network predicted by the STRING database ( https://cn.string-db.org/ ). (C) Co-IP assay was performed using an antibody specific for CUL5 to immunoprecipitate proteins from lysates of T24 and UMUC3 cells. The precipitate was subjected to western blotting with the antibodies against CUL5, PRMT5, THOC2, THRAP3, SNRNP200, PTBP1, SF3B1, PRPF8, SF3B2, and HNRNPC. (D) Western blotting with the indicated antibodies in T24 and UMUC3 cells following CUL5 knockout. (E) Co-IP assay using antibody specific for PTBP1 showed the interaction between CUL5 and PTBP1 in T24 and UMUC3 cells. The precipitate was subjected to western blotting with the antibodies against PTBP1 and CUL5. (F) Co-IP assay using antibody specific for CUL5 showed that the interaction between CUL5 and PTBP1 was not affected when bladder cancer cells were co-cultured with CD8 + T cells. The precipitate was subjected to western blotting with the antibodies against CUL5 and PTBP1. (G) Schematic diagram revealed the domains of PTBP1 full-length or truncations. (H) Co-IP assay using antibody specific for Flag showed the interaction between CUL5 and full-length or truncations of Flag-tagged recombinant PTBP1 in T24 cells. The precipitate was subjected to western blotting with the antibodies against Flag, CUL5, and β-actin. Original blots can be found in .

Article Snippet: Antibodies used included primary antibodies against CUL5 (Abclonal, A5369), β-Actin (Proteintech, 66009-1-Ig), PRMT5 (Proteintech, 18436-1-AP), THOC2 (Proteintech, 55178-1-AP), THRAP3 (Proteintech, 19744-1-AP), SNRNP200 (Proteintech, 23875-1-AP), PTBP1 (Proteintech, 12582-1-AP), SF3B1(Proteintech, 27684-1-AP), PRPF8(Proteintech, 11171-1-AP), SF3B2 (Proteintech, 10919-1-AP), HNRNPC (Proteintech, 11760-1-AP), LC3B (Abclonal, A5618), P62 (Proteintech, 18420-1-AP), Rubicon (Proteintech, 21444-1-AP), UVRAG (Proteintech, 29190-1-AP), Beclin1 (Proteintech, 11306-1-AP), HLA-class I (Proteintech, 15240-1-AP), Rabbit control IgG (Abclonal, AC005), Mouse control IgG (Abclonal, AC011), Mouse anti-HA tag (Abclonal, AE008), Rabbit anti-HA tag (Abclonal, AE036), Mouse anti-Flag tag (Abclonal, AE005), and Rabbit anti-Flag tag (Abclonal, AE004); HRP-conjugated secondary goat anti-mouse (Proteintech, SA00001-1), or goat anti-rabbit (Proteintech, SA00001-2) antibodies.

Techniques: Silver Staining, Co-Immunoprecipitation Assay, Western Blot, Knock-Out, Cell Culture, 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 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

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