Fig 2: Local translation is enriched in SN branches contacting L7 MN.A bifurcated SN was cultured with an L7 target MN and an L11 non-target MN (photomicrographs in A, D, and G) for 3 days. Alexa dye 647 (cyan) was microinjected into the SN before imaging (A, D, and G). Cultured neurons were processed for ICC for eIF4E (B; total) and 4EBP (E; total, H; phosphorylated); representative high magnification images of areas denoted by white squares in (A, D, and G) are shown in (B, E, and H), with merged DIC/Alexa fluor in the left panel, total eIF4E (B), 4EBP (E), and phospho-4EBP (H) on the right. Group data in (C, F, and I) reveal that phospho-4EBP accumulation was significantly greater in neurites contacting L7 target MNs than in neurites contacting L11 non-target MNs. However, total eIF4E (C) and total 4EBP (F) protein were distributed equally well in neurites contacting L7 target and L11 non-target MNs. Error bars represent SEM. *p < 0.05, paired t-test. Scale bars in (A), (D), and (G) =100 µm; in (B), (E), and (H) =10 µm. To test that the antibodies specifically recognized the Aplysia homologs, we performed immunoblots of Aplysia CNS lysates and of HEK cell lysate with the eIF4E, phospho-eIF4E (P-eIF4E), 4EBP, and phospho-4EBP (P-4EBP) antibodies (J).DOI: http://dx.doi.org/10.7554/eLife.04158.012

Fig 3: Ipatasertib potently suppresses 4EBP1 phosphorylation in MDA-MB-361 cells treated with combined fulvestrant and lapatinib, inhibiting newly-synthesized proteins. (a) Inhibitory effects of ipatasertib on 4EBP1 phosphorylation in fulvestrant and lapatinib-treated BT474 (left) and MDA-MB-361 (right) cells. The cells were treated with fulvestrant (100 nM), lapatinib (100 nM), ipatasertib (1000 nM), and their combinations for 24 h. Plus (+) and minus (-) indicate the presence or absence of treatment. Phosphorylated HER2, ER, and AKT were used as pharmacodynamic markers to confirm target engagement of lapatinib, fulvestrant, and ipatasertib, respectively. ß-actin was used as a loading control. Full-length blots are presented in Supplementary Figs. 8 and 9. (b) Newly synthesized protein in cells treated with DMSO, combination treatment with fulvestrant and lapatinib, and triple-combination treatment with fulvestrant, lapatinib, and ipatasertib. BT474 (left) and MDA-MB-361 (right) cells were treated with the indicated inhibitors for 24 h, and then Click-iT HPG was incorporated into drug-treated cells for 15 h. Green indicates the newly synthesized proteins detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. (c) Quantified data from Fig. 5b. The fluorescent images were captured by BZ-X800 (Keyence Corporation), and the captured images were quantified by a BZ-X800 Analyzer (Keyence Corporation). Alexa-488 fluorescence was normalized using DAPI fluorescence. The y axis indicates the relative Alexa-488 fluorescence, calculated based on fluorescence compared with the DMSO-treatment control. The data represent the mean ± SD (n = 3). The relative Alexa-488 fluorescence was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination. Differences were considered significant at p = 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. (d) Experimental schemes of cap-pulldown assay. (e) Cap pulldown assay in MDA-MB-361 cells in the triple combination. The cells were treated with fulvestrant (100 nM), lapatinib (100 nM), ipatasertib (1000 nM), and their combinations for 24 h. Immunoblotting of eIF4G, eIF4A, eIF4E, and 4EBP1 were performed in the m7 GTP pulldown samples. eIF4E was used as a pulldown positive control. Immunoblotting of eIF4G, eIF4A, eIF4E, p4EBP1, and 4EBP1 in cell lysates was performed as input controls. ß-actin was used as a loading control. Full-length blots are presented in Supplementary Fig. 10.

Fig 4: 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 5: Knockdown of eIF4E partially abrogated AEG-1-induced EMT, migration and invasion. SGC7901 and MGC803 cells were transiently transfected with AEG-1 constructs or the control vector for 24 hrs, then reseeded and transfected with eIF4E siRNA or control siRNA for another 24 hrs. Cells then underwent Western blot (A), migration (B) and invasion (C) assays. Magnification, 100×. Columns, means of five fields; bars, S.D. *P < 0.05. The data are representatives of three independent experiments.