Fig 2: Measles virus potency is reduced during inhibition of host cell translationMesothelioma cells were transfected with mismatch antisense oligonucleotide (mmASO) or eIF4E-specific ASO (4EASO) and were or were not treated with MV-CEA. H513, H2461 and H2596 were treated with MOI of 0.1 and H2373 was treated with MOI of 0.5, where indicated. H513, H2596, H2461 and H2373 were treated with 200 nM, 50 nM, 100 nM and 100 nM ASO, respectively. Following 72 hours treatment viable cells were counted. Asterisks denote a significant difference in cell viability (p<0.05) between untreated cells and the same cells treated with MV-CEA, mmASO, 4EASO or the combination treatment. (A). Mesothelioma cell lines H2373 and H513 were untreated, treated with MV-CEA at the signified MOIs or transfected with mmASO or 4EASO (200 nM for H513 and 100 nM for H2373) or the combination of agents for 48 hours. Lysates were prepared and immunoblots probed with anti-eIF4E antibody. ß-actin serves as a loading control. The normalized ratio of eIF4E to ß-actin after treatment is shown below the immunoblot. ImageJ, a public domain java image processing program was used to measure protein band intensity. (B). Human CEA production was determined by collecting medium from MM cells untreated or treated in vitro with MV-CEA, mmASO plus MV-CEA or 4EASO plus MV-CEA as in (4A). The presence of CEA was examined by ELISA technique. Standard deviation is represented by error bars. Denoted by asterisk is the significant difference in CEA levels between combination treatment of 4EASO plus MV-CEA compared to the combination treatment of mmASO plus MV-CEA or MV-CEA alone (p<0.03) (C). H2596 either expressing activated 4E-BP1A37/A46 or not were treated with the indicated MOI of MV-GFP for 72 hours and viable cells counted. Averages were compared for H2596 empty vector between treated and untreated cells by Student's t-test. * denotes a p value equal to 0.012. (D). Cell viability is expressed as cell number normalized to untreated cells. Error bars indicate standard deviation of the mean.

Fig 3: Stimulation of host cell translation enhances measles virus activityActivation of cap-dependent translation was induced by stimulation of MM cells with application of IGF-I (5 nM) followed by treatment with and without MV-GFP (MOI of 0.1) and viable cells counted 24 hours later by trypan exclusion. Cells were grown in serum free medium prior and after treatment. Columns, the mean of three independent determinations of cell viability normalized to untreated cells, bars, standard deviation. Asterisks denote a significant difference in cell survival (p<0.02) between IGF-I treated cells and the same cells infected and not infected with MV-GFP (A). MeT-5A (mesothelial) cells that produce or don't produce mouse-eIF4E (meIF4E) were treated with increasing MOI of MV-CEA. Viable cells were counted 72 hours after treatment. Data represent the mean (+/- standard deviation) of six independent determinations of cell number normalized to untreated cells. Asterisks indicate a significant difference in cell viability (p<0.012) between cells producing and not producing meIF4E treated with the same MOI of MV-CEA (B). Results from an examination of eIF4F integrity, employing a cap-affinity assay, for MeT-5A cells that produce or don't produce ectopic HA-tagged-meIF4E in cell lysates. Top, the band intensity levels for HA-meIF4E and eIF4E was determined in cell lysates derived from cells grown at steady-state. ß-actin serves as a loading control. Bottom, levels of eIF4E, HA-meIF4E and eIF4GI eluted from 7-methyl-GTP-sepahrose resin. The amount of eIF4E plus meIF4E expressed in MeT-5A cells was compared to the amount of eIF4E in empty vector cells from both lysates and for proteins (HA-meIF4E, eIF4E and eIF4GI) eluted from the cap-analogue (C). Band intensity levels were determined by using ImageJ. CEA level was assessed by collecting medium from MeT-5A cells that produce or don't produce meIF4E following treatment with or without MV-CEA (MOI 0.1). ELISA technique was used to examine the presence of CEA. Standard deviation is signified by error bars. Denoted by asterisk is the significant difference in CEA levels between MeT-5A cells that produce or don't produce meIF4E (p<0.005) (D).

Fig 4: Expression of promoters of cap-dependent translation initiation, and synaptic plasticity regulators in prefrontal cortex following 3 days active work (AW, collected at zeitgeber time, ZT0) and rest work (RW, collected at ZT12). (A,B) m7GTP pull-down analysis. (C) Representative immunoblots for (A,B). Blots normalized to total eIF4E in the corresponding immunoblot. Phospho-protein normalized to total protein in the corresponding immunoblot. (D,E) Western blot analysis. (F) Representative immunoblots for (D,E). Full-length blots/gels are presented in Supplementary Figure S2. Blots normalized to GAPDH in the corresponding immunoblots. Phospho-protein normalized to total protein in the corresponding immunoblot. Quantification of immunoblots are expressed as percentage change relative to time-matched undisturbed control (AWC collected at ZT0, RWC collected at ZT12; normalized to 100%). Plots show mean ± SEM, with scatter plot overlaid. N = 6–9/group. *p < 0.05, **p < 0.01, compared to time-matched control.

Fig 5: Profiling of signaling pathways supporting BDNF-induced Arc expression in mouse primary cortical neurons. a Western blots showing levels of Arc, phospho-p44/42 Mapk, and phospho-rpS6 in DIV13 primary cortical neurons that were treated for 6 h with a 4AP (40 µM) + Bic (50 µM) cocktail or BDNF (100 ng ml-1). Untreated cells were included as control. b–d Graphs show mean (n = 4) Arc/ß-actin (b), phospho-p42 Mapk/p42 Mapk (c), or phospho-rpS6/rpS6 (d) (±SEM) ratios for cells treated as in a. One-way analysis of variance (ANOVA) revealed a significant difference between treatment for each antigen (Arc, F 2,9 = 101.52, p < 0.0001; phospho-p42 Mapk, F 2,9 = 31.74, p < 0.0001; phospho-S6, F 2,9 = 34.49, p < 0.0001). Tukey’s HSD post hoc test, *p < 0.05; **p < 0.01; ****p < 0.0001. e Western blot of Arc protein expression in cortical neurons co-treated for 6 h with BDNF plus vehicle (DMSO) or Mek/Mapk pathway inhibitor U0126 (10 µM). f Similar experiment as in e was conducted with Mnk1 inhibitor CGP 57380 (5 µM) or Rac1 inhibitor NSC23766 (200 µM). Experiments in e, f were done in duplicate and lysates were probed for phospho-eIF4E, phospho-rpS6, and/or phospho-p44/42 Mapk (g) as control. h Arc mRNA expression in cortical neurons treated for 6 h with BDNF plus vehicle, U0126, or NSC23766. Inhibition of Mek/Mapk pathway by U0126 prevented BDNF-induced Arc expression but not Rac1 inhibition by NSC23766. Map2 expression was assessed as control. Bars represent mean fold-change measured by RT-qPCR (error bars indicate range from three biological replicates). i Schema summarizing the contribution of Mek/Mapk and Rac1/rpS6 signaling to BDNF-induced Arc expression in primary cortical neurons