Journal: Molecules and Cells

Article Title: Combination Therapy with a PI3K/mTOR Dual Inhibitor and Chloroquine Enhances Synergistic Apoptotic Cell Death in Epstein–Barr Virus-Infected Gastric Cancer Cells

doi: 10.14348/molcells.2019.2395

Figure Lengend Snippet: (A) Representative images of EBV- and mock-infected AGS cells 48 hours post-sequential combination therapy (SCT; cells were incubated with 10 μM CQ for 1 hour followed by concomitant 10 μM CQ and 100 nM of CMG002 for the remainder of the 48-hour period) (40× magnification). (B) Flow cytometry-based TUNEL assay demonstrating gating of apoptotic cells 48 hours following monotherapy and SCT. The percentage of apoptotic cells represents the mean of more than three independent experiments. (C) Apoptotic index (%) in EBV- and mock-infected AGS cells after 48 hours of treatment with CQ and CMG002 monotherapy and SCT. Results expressed represent the mean ± SEM from experiments performed in triplicate. (D) Western blot analysis of the apoptotic marker proteins, caspase-3 and PARP-1, and the autophagy marker LC3B. Western blot data represent experiments performed in triplicate. * P < 0.05.

Article Snippet: After blocking, the membranes were subjected to western blotting with primary antibodies against Epstein–Barr nuclear antigen1 (EBNA1) (#sc-81581; Santa Cruz Biotechnology, USA); poly (ADP-ribose) polymerase 1 (PARP-1) (#sc-7150; Santa Cruz Biotechnology); β-actin (#sc-47778; Santa Cruz Biotechnology); Akt (#9272s; Cell Signaling Technology [CST], USA); phospho-Akt (Ser473) (#9271s; CST); extracellular signal–regulated kinase1/2 (Erk1/2) (#4695s; CST); phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370s; CST); S6 ribosomal protein (#2217s; CST); cleaved caspase-3 (#9664; CST); caspase-3 (#9662; CST); light chain 3B (LC3B) (#2775; CST); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#97166; CST) incubated at 4°C overnight.

Techniques: Infection, Incubation, Flow Cytometry, TUNEL Assay, Western Blot, Marker

Journal: Molecules and Cells

Article Title: Combination Therapy with a PI3K/mTOR Dual Inhibitor and Chloroquine Enhances Synergistic Apoptotic Cell Death in Epstein–Barr Virus-Infected Gastric Cancer Cells

doi: 10.14348/molcells.2019.2395

Figure Lengend Snippet: (A) Representative images of EBV- and mock-infected NUGC3 cells 48 hours post-SCT with CQ and CMG002 (40× magnification). (B) Flow cytometry-based TUNEL assay demonstrating gating of apoptotic cells 48 hours following monotherapy and SCT. The percentages of apoptotic cells represent the mean of more than three independent experiments. (C) Apoptotic index (%) in EBV- and mock-infected NUGC3 cells after 48 hours of treatment with monotherapy and SCT. Results expressed represent the mean ± SEM from the experiments conducted in triplicate. (D) Western blot analysis of the apoptotic marker proteins, caspase-3 and PARP-1, and the autophagy marker LC3B. Western blot data represent experiments performed in triplicate. * P < 0.05.

Article Snippet: After blocking, the membranes were subjected to western blotting with primary antibodies against Epstein–Barr nuclear antigen1 (EBNA1) (#sc-81581; Santa Cruz Biotechnology, USA); poly (ADP-ribose) polymerase 1 (PARP-1) (#sc-7150; Santa Cruz Biotechnology); β-actin (#sc-47778; Santa Cruz Biotechnology); Akt (#9272s; Cell Signaling Technology [CST], USA); phospho-Akt (Ser473) (#9271s; CST); extracellular signal–regulated kinase1/2 (Erk1/2) (#4695s; CST); phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370s; CST); S6 ribosomal protein (#2217s; CST); cleaved caspase-3 (#9664; CST); caspase-3 (#9662; CST); light chain 3B (LC3B) (#2775; CST); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#97166; CST) incubated at 4°C overnight.

Techniques: Infection, Flow Cytometry, TUNEL Assay, Western Blot, Marker

Journal: Journal of Virology

Article Title: B Cell-Specific Transcription Activator PAX5 Recruits p300 To Support EBNA1-Driven Transcription

doi: 10.1128/JVI.02028-19

Figure Lengend Snippet: Antibodies and primers used in this study

Article Snippet: EBNA1-FITC , Biorbyt , orb4774.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: EBV infection suppresses TLR9 m 6 A modification and expression. A , schematic diagram of the MeRIP-seq protocol used to identify RNA m 6 A profile changes in BJAB cells upon EBV infection. B , GO and KEGG pathway enrichment results for downregulated m 6 A-modified genes upon EBV infection ( p ≤ 0.05, date from our previous study: #GSE133936). C , BJAB cells were infected with 50 MOI EBV for 48 h. TLR9, EBNA1, and GAPDH protein expressions were examined. D , m 6 A -RIP-qPCR analysis of TLR9 mRNA expression in BJAB cells upon EBV infection. Control group was used for negative control. E and F , Flag-TLR9 was pretransfected into HEK293 cells for 6 h, then, Flag-NC, Flag-EBNA1, or Flag-EBNA1△NLS plasmids were respectively transfected for 24 h. The mRNA and protein expressions were measured by RT-qPCR and Western blotting ( E ). The m 6 A abundance of TLR9 mRNA was analyzed by m 6 A -RIP-qPCR (F). Three independent experiments were performed, and data are shown as the mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; m 6 A, N 6 -methyladenosine; MeRIP, methylated RNA immunoprecipitation; MOI, multiplicity of infection; TLR, toll-like receptor; RIP, RNA immunoprecipitation.

Article Snippet: EBNA1 plasmid is purchased from Addgene (#37954).

Techniques: Infection, Modification, Expressing, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Virus, Methylation, RNA Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: TLR9 downstream signaling pathways and cytokine expression are regulated by METTL3. A , cellular m 6 A modification levels were assayed in BJAB-shNC and BJAB-shMETTL3 cells by Dot blot analysis. B , the expression levels of TLR9 and METTL3 were determined by Western blotting in BJAB-shNC and BJAB-shMETTL3 cells. C , BJAB-shNC and BJAB-shMETTL3 cells were treated with ActD (5 μg/ml) for 0, 3, and 6 h; TLR9 mRNA levels were measured by RT-qPCR. D , the indicated TLR9 signaling proteins were detected by cytoplasmic and nuclear proteins isolation. GAPDH and Histone H3 were used for cytoplasm and nucleus protein loading controls, respectively. E , BJAB cells were treated with STM2457(10 μM) and CpG-ODN2006 (5 μM) upon 50 MOI EBV infection, respectively. EBV genomes copy number was determined by qPCR. F , primary B cells were treated with STM2457(10 μM) and CpG-ODN2006(5 μM) with or without 50MOI EBV infection for appointed time. The secretion of IFN-α and CXCL10 were assayed by ELISA. These data are shown as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; IFN, interferon; m 6 A, N 6 -methyladenosine; MOI, multiplicity of infection; ODN, oligodeoxynucleotide; RT-qPCR, reverse transcription-quantitative PCR; TLR9, toll-like receptor 9.

Article Snippet: EBNA1 plasmid is purchased from Addgene (#37954).

Techniques: Protein-Protein interactions, Expressing, Modification, Dot Blot, Western Blot, Quantitative RT-PCR, Isolation, Infection, Enzyme-linked Immunosorbent Assay, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: EBNA1 induces METTL3 protein degradation via the ubiquitination pathway. A , EBNA1 protein levels were detected in BJAB, Raji, and B95.8 cells. B and C , Flag-NC and Flag-EBNA1 were transfected into HEK293 cells. The mRNA and protein levels of METTL3 and EBNA1 were assayed by RT-qPCR ( B ) and Western blotting ( C ). D , The 100 ng, 200 ng, and 500 ng mRNA from HEK293 cells transfected with Flag-EBNA1 (or Flag-NC as negative control) were extracted to detect the total cellular m 6 A modification level by Dot blot. A concentration of 0.02% methylene blue staining was used as a loading control. E and F , HEK293 cells were transfected with EBNA1 plasmids for 36 h, followed by CHX treatment (25 μg/ml) and MG132 (25 μM) for indicated times. Then cellular proteins were collected, and Western blotting was performed. G , GFP-NC and GFP-EBNA1, Flag-METTL3 and plasmids containing various polyubiquitin chains were cotransfected for 48 h. HEK293 cells were treated with MG132 (25 μM) for 4 h before harvest. Lysates were immunoprecipitated with anti-Flag beads. H , HONE-1 cells were transfected with indicated siRNAs for 48 h. The protein expression levels were detected by Western blotting. I and J , HONE-1 cells were transfected with indicated plasmids, then, the mRNA and protein expression of PRKN was measured by RT-qPCR ( I ) and Western blotting ( J ). K , HEK293 cells were transfected with Flag-PRKN and Myc-METTL3 plasmids for 48 h. The cell lysates were immunoprecipitated with anti-Flag beads (or IgG as control). The first lane was an input control without immunoprecipitation. L , Myc-METTL3, Flag-PRNK, and Ub (either WT or K48R) plasmids were cotransfected into HEK293 cells for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibodies. The data are shown as the mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. CHX, cycloheximide; EBNA1, Epstein-Barr nuclear antigen 1; m 6 A, N 6 -methyladenosine; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: EBNA1 plasmid is purchased from Addgene (#37954).

Techniques: Ubiquitin Proteomics, Transfection, Quantitative RT-PCR, Western Blot, Negative Control, Modification, Dot Blot, Concentration Assay, Staining, Control, Immunoprecipitation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: A working model depicting the main molecular mechanisms about the current study. EBV EBNA1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway, which mediated by the E3 ligase PRKN, thus inhibits cellular m 6 A modification. Downregulation of METTL3 inhibits TLR9 m 6 A modification. YTHDF1 is an m 6 A “reader” for cellular TLR9 mRNA with m 6 A modification, and YTHDF1 binding increases TLR9 mRNA translation efficiency. EBV infection decreases the m 6 A levels of TLR9 mRNA, thus reduces TLR9 protein levels, as well as TLR9-induced cytokine secretions. EBV, Epstein-Barr virus; m 6 A, N 6 -methyladenosine; TLR9, toll-like receptor 9.

Article Snippet: EBNA1 plasmid is purchased from Addgene (#37954).

Techniques: Ubiquitin Proteomics, Modification, Binding Assay, Infection, Virus

Journal:

Article Title: AMP-Activated Protein Kinase Deficiency Exacerbates Aging-Induced Myocardial Contractile Dysfunction

doi: 10.1111/j.1474-9726.2010.00586.x

Figure Lengend Snippet: Western blot analysis depicting protein expression of ryanodine receptor (RyR), L-type Ca2+ channel dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), Na+/Ca2+ exchanger (NCX), phospholamban (PLB) and phosphorylated PLB (pPLB, Ser16) in myocardium from young or old wild-type (WT) and AMPK deficient KD mice. (A) Representative gel blots of RyR, DHPR, SERCA2a, NCX, PLB, pPLB and GAPDH (loading control) using specific antibodies; (B) RyR expression; (C) DHPR expression; (D) SERCA2a expression; (E) NCX expression; (F) PLB expression; (G) pPLB (Ser16) expression and (H) pPLB-to-PLB ratio. Mean ± SEM, n = 3 – 6 mice per group, *p < 0.05 vs. respective young group, #p < 0.05 vs. WT-old group.

Article Snippet: Membranes were probed with anti-Akt (1:1,000), anti-pAkt (Thr 473 , 1:1000), anti-AMPK (1:1,000), anti-pACC (Ser 79 , 1:1,000), anti-HSP90 (1:1,000 ), anti-peNOS (Ser 1177 , 1:1,000), anti-Glut4 (1:1,000, all from Cell Signaling), anti-PGC-1α (1:500) anti-DHPR (1:200), anti-RyR (1:100, all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-SERCA2a (1:1,000, Bethyl Laboratories Inc., Montgomery, TX), anti-PLB (1:1,000), anti-pPLB (Ser 16 , 1:1000 dilution; both from Upstate, Lake Placid, NY), anti- Na + /Ca 2+ exchanger (Abcam Inc., Cambridge, MA) and anti-GAPDH (1:1,000, internal loading control, Cell Signaling) antibodies.

Techniques: Western Blot, Expressing