Journal: PLoS ONE

Article Title: MNK1-Induced eIF-4E Phosphorylation in Myeloma Cells: A Pathway Mediating IL-6-Induced Expansion and Expression of Genes Involved in Metabolic and Proteotoxic Responses

doi: 10.1371/journal.pone.0094011

Figure Lengend Snippet: A ) MM lines exposed to IL-6 (100 U/ml) for 3 hrs followed by immunoblot assay for phospho-MNK and total MNK expression. Fold increase is determined by densitometric ratio of MNK-P/MNK-total and represents the mean of 3 independent experiments. * denotes IL-6-induced increase is statistically significant (p<0.05). B ) ANBL-6 cells exposed to IL-6 for 0, 60, 120 or 180 mins, followed by immunoblot assay. C ) Primary cells from 4 patients exposed to IL-6 or media (C) followed by immunoblot assay. D ) ANBL-6 cells treated with IL-6 for increasing durations, followed by immunoprecipitation with anti-MNK1, MNK2 or control IgG. Immunoprecipitates tested for phosphorylation of eIF-4E in vitro.

Article Snippet: The HA-tagged eIF-4E full length coding sequence was isolated from pHA-eIF4E (obtained from Addgene, plasmid 17343) by PCR.

Techniques: Western Blot, Expressing, Immunoprecipitation, In Vitro

Journal: PLoS ONE

Article Title: MNK1-Induced eIF-4E Phosphorylation in Myeloma Cells: A Pathway Mediating IL-6-Induced Expansion and Expression of Genes Involved in Metabolic and Proteotoxic Responses

doi: 10.1371/journal.pone.0094011

Figure Lengend Snippet: A ) ANBL-6 or 8226 cells transfected with empty vector (EV), serine-to-alanine phosphodefective eIF-4E (SA), serine-to-aspartic acid phosphomimetic eIF-4E (ASP) or wild type (WT) eIF-4E, followed by immunoblot assay. Transfected (transfex) vs endogenous (endog) eIF-4E denoted. B ) Above described ANBL-6 and 8226 transfected cell lines treated +/− IL-6 (100 U/ml) for varied durations and viable cell recovery enumerated. Results are relative cell growth compared to number of cells present at time 0 (mean+/−SD (n = 3)). * denotes statistically significant (p<0.05) alteration in cell recovery versus empty vector-transfected cells.

Article Snippet: The HA-tagged eIF-4E full length coding sequence was isolated from pHA-eIF4E (obtained from Addgene, plasmid 17343) by PCR.

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: MNK1-Induced eIF-4E Phosphorylation in Myeloma Cells: A Pathway Mediating IL-6-Induced Expansion and Expression of Genes Involved in Metabolic and Proteotoxic Responses

doi: 10.1371/journal.pone.0094011

Figure Lengend Snippet: A ) 8226 cells injected SQ and mice randomized to control (C) group, low dose CGP57380 (LD, 20 mg/kg) or high dose (HD, 50 mg/kg). Treatment is daily IP injections. At day +4 and +7 of treatment, tumors were measured. Data is tumor volume, mean+/−SD (n = 8). * = significantly (p<0.05) smaller tumor vs control. B and C ) Individual control (C), high dose (HD)-treated or low dose (LD)-treated tumors harvested after 3 days of treatment for Western blot, assaying phospho-eIF-4E, total eIF-4E, GAPDH and PARP that has been cleaved by caspase (AB #954 from Cell Signaling). D ) Mice challenged SQ with 8226 cells transfected with EV (right flank) and 8226 cells transfected with dominant negative eIF-4E (left flank). A separate cohort of mice were challenged with non-transfected parental 8226 cells. Tumor growth is mean+/−SD tumor volume, n = 8.

Article Snippet: The HA-tagged eIF-4E full length coding sequence was isolated from pHA-eIF4E (obtained from Addgene, plasmid 17343) by PCR.

Techniques: Injection, Western Blot, Transfection, Dominant Negative Mutation

Journal: PLoS ONE

Article Title: MNK1-Induced eIF-4E Phosphorylation in Myeloma Cells: A Pathway Mediating IL-6-Induced Expansion and Expression of Genes Involved in Metabolic and Proteotoxic Responses

doi: 10.1371/journal.pone.0094011

Figure Lengend Snippet: A ) AHA incorporation is tested in MM cells stimulated with IL-6 for 30, 60, 120 or 180 mins. In B ), AHA incorporation is tested in IL-6-stimulated EV-, WT or mutant SA eIF-4E-transfected MM cells. In C ), EV or SA mutant-expressing cells are pre-treated +/− pp242, followed by IL-6. Bar graphs under panels are means+/−SD of 4 separate experiments. * denotes statistically significant increase in AHA incorporation induced by IL-6 in A and B and decrease versus IL-6-stimulated control in C). D ) Lambda light chain ELISA assay in MM cell lysates, comparing EV- vs mutant eIF-4E-transfected cells +/− IL-6 and +/− pp242. Data are means+/−SD, n = 4. * denotes statistically significant difference from corresponding controls.

Article Snippet: The HA-tagged eIF-4E full length coding sequence was isolated from pHA-eIF4E (obtained from Addgene, plasmid 17343) by PCR.

Techniques: Mutagenesis, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: MNK1-Induced eIF-4E Phosphorylation in Myeloma Cells: A Pathway Mediating IL-6-Induced Expansion and Expression of Genes Involved in Metabolic and Proteotoxic Responses

doi: 10.1371/journal.pone.0094011

Figure Lengend Snippet: A ) Different categories of 166 genes demonstrating significantly inhibited translation in mutant-expressing cells. B ) Immunoblot analysis of protein expression in EV-, WT and mutant (MU)-expressing MM cells. C ) Immunoblot analysis of protein expression in EV vs mutant (MU) eIF-4E-expressing MM cells +/− IL-6 for 48 hrs.

Article Snippet: The HA-tagged eIF-4E full length coding sequence was isolated from pHA-eIF4E (obtained from Addgene, plasmid 17343) by PCR.

Techniques: Mutagenesis, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance *

doi: 10.1074/jbc.M111.304626

Figure Lengend Snippet: Effects on 4E-BP1/eIF-4E mediate ERK activation. In A, 8226 or MM1.S cells treated with IGF-1 (200 ng/ml) ± rapamycin or pp242 for 30 min followed by eIF-4E precipitation and subsequent immunoblot of the precipitate for eIF-4E, 4E-BP1, and eIF-4G presence. In B, similar experiment performed in 8226 cells without presence of IGF-1. In C, empty vector (EV) or HA-eIF-4E- stably transfected MM1.S cells (left panel) or 8226 cells (right panel) treated with increasing concentrations of pp242 followed by immunoblot assay for phospho-ERK, total ERK, and HA tag. In D, empty vector or HA-eIF-4E transiently transfected 8226 cells treated with increasing concentrations of pp242 followed by immunoblot assay for phosphorylated and total ERK or phosphorylated and total MEK. In E, empty vector or eIF-4E-transfected cells treated with increasing concentrations of pp242 followed by immunoprecipitation of c-RAF and assay for raf in vitro kinase activity against MEK substrate. Fold increase in kinase activity calculated as described under “Experimental Procedures.”

Article Snippet: The HA-tagged eif4e full-length coding sequence was isolated from pHA-eif4e (obtained from Addgene, plasmid 17343) by PCR, using primers Spe1HA-eif4e (5-AAAGACTAGTATGTACCCATACGACGTCCC-3) and Xho1HA-eif4e (5-GATGCATGCTCGAGTTAAACA-3) to produce 5′-Spe1HA-eif4eXho1–3′ PCR product.

Techniques: Activation Assay, Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Immunoprecipitation, In Vitro, Activity Assay