Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Measles vaccine strains for virotherapy of non-small cell lung carcinoma

doi: 10.1097/JTO.0000000000000214

Figure Lengend Snippet: A) H2009, A549, and H838 cell lysates were assayed by immunoblot before and 48 hours after infection with MV-GFP for key proteins involved in cap-dependent and independent translation initiation. B) Beas2B expressing empty vector (Beas2B-V) and Beas2B expressing eIF4E (Beas2B-E) were infected with MV-CEA at indicated MOI and assayed for cell viability 72 hours after infection. 5’ cap-affinity assay of untreated Beas2B-V and Beas2B-E are shown. Relative increases in eIF4G binding in the cap-affinity assay correspond to enhanced eIF4F cap-complex formation. C) H2009 and H522 NSCLC cells were treated with MV-CEA (MOI=0.01), rapamycin (Rap), or the combination and assayed for cell viability 72 hours after infection. Supernatants were collected from cells and assayed for CEA production as a measure of viral replication. Error bars indicate standard deviation of the mean. * indicates statistical significance. D) H2009 and H522 cells were treated with MV-CEA (MOI=0.01), 4EGI-1 (15μM), or the combination and cell viability assayed 72 hours after infection. E) NSCLC cell lines were treated with MV-GFP at MOI of 0.25 either alone or in combination with rapamycin. Representative fluorescence micrographs are shown.

Article Snippet: The primary antibodies employed were rabbit α-eIF4E, rabbit α-4E-BP1, rabbit α-PARP, α-eIF2α, α-phospho-eIF2α(Ser 51 ), α-PKR, α-phospho-PKR, all from Cell Signaling at a 1:1000 dilution, mouse α-b-actin (Sigma) at a 1:10,000 dilution and rabbit α-eIF4GI (kindly provided by Nahum Sonenberg, McGill University Montreal, Quebec, Canada) at a 1:2500 dilution.

Techniques: Western Blot, Infection, Expressing, Plasmid Preparation, Binding Assay, Standard Deviation, Fluorescence

Journal: Molecules and Cells

Article Title: OASL1 Traps Viral RNAs in Stress Granules to Promote Antiviral Responses

doi: 10.14348/molcells.2018.2293

Figure Lengend Snippet: (A) OASL1 associated with cytoplasmic speckles specifically upon poly(I:C) treatment. NIH-3T3 cells expressing EGFP-OASL1 are shown following stimulation with various TLRs (indicated at the top) for 6 h (top panel) or 12 h (lower panel). (B) OASL2 did not associate with speckles upon poly(I:C) stimulation. EGFP-OASL2–overexpressing NIH-3T3 cells were stimulated with poly(I:C) for 6 h. (C) OASL1-containing speckles do not overlap with secretory vesicles. EGFP-OASL1–overexpressing NIH-3T3 cells were stained with specific antibodies against the indicated markers (KDEL, RCAS, LAMP1, and PMP70; shown in red). (D) OASL1-containing speckles colocalized with SG markers. EGFP-OASL1–expressing cells were stained with the indicated SG markers (TIAR, S6, eIF4E, and G3BP1) after stimulation with poly(I:C) for 6 h. (E) Actin and microtubules are required for formation of OASL1-containing speckles. EGFP-OASL1–xpressing cells were stained for β-actin after pre-treatment with Cytochalasin D or colchicine treatment followed by poly(I:C) stimulation. Nuclei were stained with DAPI (blue). Scale bars correspond to 10 μm. Images are representative of at least two independent experiments.

Article Snippet: The membranes were blocked with 5% w/v skim milk in TBS with 0.1% Tween-20 (TBST) for 1 h at RT, and then probed with primary antibodies diluted in blocking solution: rabbit α-OASL1 , rabbit α-PKR (Santa Cruz), mouse α-HSP70 (Enzo Life Sciences), rabbit α-eIF4E (Cell Signaling), rabbit α-IRF7 (Invitrogen), or rabbit α-GAPDH (Santa Cruz).

Techniques: Expressing, Staining

Journal: Nature

Article Title: mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis

doi: 10.1038/s41586-018-0538-8

Figure Lengend Snippet: a, EM images of polyribosome with METTL3-gold particle labeling. Red arrows indicate METTL3 with immuno-gold particle (6 nm). Three independently performed experiments show similar results. b, Counting of METTL3 with gold particle labeling in each polyribosome. c, EM images of polyribosome with METTL3 and eIF4E. Red arrows indicate METTL3 with immuno-gold particle (6 nm) and yellow arrows indicate eIF4E with immuno-gold particle (10 nm). Four independently performed experiments show similar results. d, Average distance between immuno-gold particles was measured. n = 6 biologically independent samples from at least three independent experiments. Error bars represent mean ± SD. e, Colloidal Coomassie blue staining of recombinant protein His-METTL3 or His-METTL3 1-200 amino acid fragments (1-200). Two independently performed experiments show similar results. f, Colloidal Coomassie blue staining of recombinant GST-tagged protein eIF3g, eIF3h, eIF3i, eIF3j or eIF3m. Two independently performed experiments show similar results. g, GST-eIF3h was co-purified with His-METTL3 in the presence of either rabbit IgG (rIgG) or α-METTL3 antibody. Levels of co-purified His-METTL3 were analyzed by Western blotting. Two independently performed experiments show similar results. h, Schematic diagram of human eIF3h deletion mutants. i, Colloidal Coomassie blue staining of recombinant GST-eIF3h, -eIF3h (1-222) or -eIF3h (29-222). n = 1 independent experiments. j, GST pull-down of indicated eIF3h deletion mutants. Co-purified His-METTL3 was analyzed by Western blotting. n = 1 independent experiments. k, Western blotting demonstrates efficient knockdown of eIF3h protein. Three independently performed experiments show similar results. l, qRT-PCR analysis demonstrates efficient down regulation of eIF3h mRNA. Error bars represent mean ± SD; n = 3 biologically independent samples; two-sided t-test. m, qRT-PCR analysis of reporter mRNAs. FLuc-MS2bs reporter mRNAs were normalized to RLuc mRNAs. The FLuc:RLuc ratio obtained in FLAG-MS2 was set to 1. Error bars represent mean ± SD; n = 3 biologically independent samples.

Article Snippet: Where indicated, during the elution, α-METTL3 antibody (Proteintech, 15073-1-AP) and gold nanoparticle (6nm) conjugated α-rabbit IgG were added with/without either α-CBP80 antibody or α-eIF4E antibody that was gold nanoparticle (10nm) conjugated using GOLD conjugation kit (Abcam, ab201808) according to the manufacturer’s instructions.

Techniques: Labeling, Staining, Recombinant, Purification, Western Blot, Quantitative RT-PCR

Journal: British Journal of Cancer

Article Title: Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma

doi: 10.1038/sj.bjc.6605184

Figure Lengend Snippet: IGF-I stimulation activates downstream-signalling pathways and inactivates 4E-BP1 in mesothelioma cell lines. ( A ) Immunoblot analysis showing phosphorylation and total levels of proteins involved in cell-signalling pathways (IGF1R, Akt, and MAPK) or in initiation of translation (eIF4G, eIF4E, and 4E-BP1) after treatment (in minutes) with and without IGF-I (5 n M ) or on treatment with IGF-I (5 n M for 20 min) combined with LY249002 (LY) or U0126 (U). ( B ) Representation of the percentage of 4E-BP1 in hypophosphorylated isoforms (α, open columns) compared with that in hyperphosphorylated isoforms (β + γ, filled columns) for mesothelioma cells and mesothelial control cells (LP9) not treated or treated with IGF-I (5 n M ) or IGF-I combined with LY249002 (LY) or U0126 (U) for the indicated times. β-actin represents loading controls for each cell line.

Article Snippet: Blots were probed separately with either rabbit α-eIF4GI antibody (kindly provided by Nahum Sonenberg, McGill University Montreal, Quebec, Canada) at a 1 : 2500 dilution, rabbit α-IGF1R antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a 1 : 1000 dilution, rabbit α-phospho-IGF1R antibody (Cell Signaling, Danvers, MA, USA) at a 1 : 1000 dilution, rabbit α-Akt antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-phospho-Akt (ser473) antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-MAPK antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-phospho-MAPK (Thr202/Tyr204) antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-4E-BP1 antibody (Abcam Inc., Cambridge, MA, USA) at a 1 : 2500 dilution, α-phospho-4E-BP1 (ser65) antibody (Cell Signaling) at a 1 : 1000 dilution, α-eIF4E antibody (BD Biosciences, San Jose, CA, USA) at a 1 : 500 dilution, mouse α-actin (Sigma) at a 1 : 10 000 dilution, or rat α-HA antibody (Roche) at a 1 : 2000 dilution to detect the haemagglutinin-tagged HA-4E-BP1 A37/A46 proteins.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma

doi: 10.1038/sj.bjc.6605184

Figure Lengend Snippet: IGF-I stimulation activates cap-mediated translation in mesothelioma. ( A ) After overnight serum starvation, cells were treated or not treated with IGF-I (5 n M ) for the indicated times (minutes) or treated with IGF-I combined with LY249002 (LY). Samples were subjected to cap-analogue capture using 7-methyl-GTP-sepharose before immunoblot analysis. ( B ) Depiction of the relative level of eIF4G normalised to eIF4E bound to the cap analogue for each cell line.

Article Snippet: Blots were probed separately with either rabbit α-eIF4GI antibody (kindly provided by Nahum Sonenberg, McGill University Montreal, Quebec, Canada) at a 1 : 2500 dilution, rabbit α-IGF1R antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a 1 : 1000 dilution, rabbit α-phospho-IGF1R antibody (Cell Signaling, Danvers, MA, USA) at a 1 : 1000 dilution, rabbit α-Akt antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-phospho-Akt (ser473) antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-MAPK antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-phospho-MAPK (Thr202/Tyr204) antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-4E-BP1 antibody (Abcam Inc., Cambridge, MA, USA) at a 1 : 2500 dilution, α-phospho-4E-BP1 (ser65) antibody (Cell Signaling) at a 1 : 1000 dilution, α-eIF4E antibody (BD Biosciences, San Jose, CA, USA) at a 1 : 500 dilution, mouse α-actin (Sigma) at a 1 : 10 000 dilution, or rat α-HA antibody (Roche) at a 1 : 2000 dilution to detect the haemagglutinin-tagged HA-4E-BP1 A37/A46 proteins.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma

doi: 10.1038/sj.bjc.6605184

Figure Lengend Snippet: Assembly of cap-dependent initiation complex in mesothelioma is impaired by the production of constitutively active 4E-BP1 (4E-BP1 A37/A46 ). ( A ) An assessment of eIF4F integrity utilising a cap-affinity assay of four mesothelioma cell lines ectopically expressing the 4E-BP1 A37/A46 protein or control. Top, steady state levels of exogenous HA-tagged 4E-BP1 A37/A46 and endogenous 4E-BP1 in cells. Bottom , eIF4E and binding partners eIF4G and 4E-BP1 A37/A46 eluted from 7-methyl-GTP-sepharose resin. ( B ) The average and s.d. for the 4 MM cell lines, relative level of eIF4G normalised to eIF4E bound to the cap analogue comparing cells expressing and not expressing 4E-BP1 A37/A46 .

Article Snippet: Blots were probed separately with either rabbit α-eIF4GI antibody (kindly provided by Nahum Sonenberg, McGill University Montreal, Quebec, Canada) at a 1 : 2500 dilution, rabbit α-IGF1R antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a 1 : 1000 dilution, rabbit α-phospho-IGF1R antibody (Cell Signaling, Danvers, MA, USA) at a 1 : 1000 dilution, rabbit α-Akt antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-phospho-Akt (ser473) antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-MAPK antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-phospho-MAPK (Thr202/Tyr204) antibody (Cell Signaling) at a 1 : 1000 dilution, rabbit α-4E-BP1 antibody (Abcam Inc., Cambridge, MA, USA) at a 1 : 2500 dilution, α-phospho-4E-BP1 (ser65) antibody (Cell Signaling) at a 1 : 1000 dilution, α-eIF4E antibody (BD Biosciences, San Jose, CA, USA) at a 1 : 500 dilution, mouse α-actin (Sigma) at a 1 : 10 000 dilution, or rat α-HA antibody (Roche) at a 1 : 2000 dilution to detect the haemagglutinin-tagged HA-4E-BP1 A37/A46 proteins.

Techniques: Expressing, Binding Assay