Figure 1

eIF4A inhibition reveals genes under translational control in MDA-MB-231 cells. (a) MDA-MB-231 breast cancer cells or (b) MCF-10A non-malignant luminal breast cells were treated with increasing concentrations of Silvestrol for 72 hours. Cell proliferation was measured by lysing in Cell Titer Glow reagent and measuring luminescence in relative light units (RLU EC₅₀ MDA-MB-231 = 6 nM; EC₅₀ MCF-10A >3 μM. (c) MDA-MB-231 cells were treated with DMSO or 25 nM Silvestrol, pulsed with ³⁵S-methionine for 15 minutes prior to harvest and harvested at 0, 1 or 2 hours after treatment. Bar graph represents counts per minute (CPM) normalized to total protein. Error bars represent standard error (n = 3). (d) Polysome profiles of MDA-MB-231 cells treated with DMSO (top panel; 21% ribosomal subunits and 80S monosomes, 79% polysomes; n =2) or with 25 nM Silvestrol (bottom panel; 75% ribosomal subunits and 80S monosomes, 25% polysomes; n =2) for 2 hours. A₂₆₀, the absorbance of light at 260 nm. (e) Distribution of ribosome footprint (RF) RPKM values from DMSO-treated MDA-MB-231 cells (mean Log₂ RPKM value =4.78) compared with Silvestrol-treated cells (mean Log₂ RPKM value =4.49). RPKM = reads per kilobase per million. (f) Scatter plot of RF densities (measured in RPKM) in cells treated with 25 nM Silvestrol versus DMSO for 1 or 2 hours. Silvestrol-sensitive genes are indicated in dark blue. Correlation coefficients for biological replicates (n =2) are DMSO r =0.945 and Silvestrol r =0.977. (g) Distribution of changes in translation efficiency (TE) between DMSO- or Silvestrol-treated cells. To calculate TE, RPKM values from RF RNAs were normalized to RPKM values from mRNA sequencing results generated from identical biological samples. Silvestrol-sensitive genes with decreased TE (z-score below -1.5) and Silvestrol-resistant genes (z-score >1.5) are indicated. Population mean indicated by a solid vertical line; dotted vertical lines indicate σ values above and below the mean.