Fig 1: eIF4A regulates ER activity and cell growth(A) MCF7 treated for 24hr with 20nM Silvestrol followed by analysis of canonical ER target gene expression by RT-qPCR. P-values were determined by students t-test for each gene. P< .01 depicted as ** and p< .001 as ***. Data is representative of three independent experiments.(B) MCF7 were plated in DMEM F12 containing charcoal stripped FBS and lacking phenol red, followed by treatment with silvestrol (20nM) for 24hr. Cells were then stimulated with 10nM estradiol for an additional 24hr. PGR mRNA expression was analyzed by RT-qPCR. P-values were determined by ordinary one-way ANOVA. P< 0.001 is depicted as ***. Data is representative of three independent experiments.(C) MCF7 were placed in DMEM F12 containing charcoal stripped FBS and lacking phenol red, followed by treatment with 20nM Silvestrol for 24hr. Cells were then stimulated with 10nM estradiol for 1hr. ER binding to the TFF1 enhancer element was analyzed by Chromatin Immunoprecipitation Assay (ChIP) and quantified by RT-qPCR. Representative data is shown with technical replicates for n=3. P-values were determined by ordinary one-way ANOVA. P< 0.001 is depicted as ***. Data is representative of three independent experiments.(D) MCF7 cells were plated in 96 well plates and treated for up to seven days with increasing doses of silvestrol. Cell viability was measured at each timepoint using ATP-glo luminescence. Data is representative of three independent experiments.(E) T47D cells were plated in 96 well plates and treated for up to seven days with increasing doses of silvestrol Cell viability was measured at each timepoint using ATP-glo luminescence. Data is representative of three independent experiments.(F) MCF7 were treated with Veh. (DMSO) or silvestrol (20nM) in triplicate for 24hr. followed by analysis of protein expression by tandem mass tag LC-MS. Significant hits were those proteins changing by at least log2 fold change>1 and an adj. P-value of <.05 (see methods).(G) MCF7 were treated with 20nM Silvestrol for the indicated times. Data is representative of three independent experiments.