(a) Binding site of oligonucleotides 4 (red) and 5.4 (blue) in the ES6S region of the 40S subunit (left panel). Note that in this model of PIC (EMD-5658), the ES6SA helix is in an ‘outward’ orientation. Right panels show VIC-oligo 4 uptake and binding to ES6SD. The upper panel shows fluorescence microscopy of VIC-oligo 4 (green) combined with an IF staining of eS7 (red) and DAPI (blue) in MEF cells. Note the typical aggregation of the oligo inside the cells. The bottom panel shows an in situ RT-PCR of RNA extracted from the indicated fractions. cDNA was primed exclusively by the VIC-oligo 4, which remained bound to ES6SD during RNA extraction. Total RNA from untransfected cells was included as a negative control. (b) Metabolic labeling of MEF cells with [35S]-Met. Cells transfected with the indicated oligonucleotides were labeled for 30 min at 12 hr post-transfection (hpt), and analyzed by SDS-PAGE and autoradiography. A SYPRO staining of the middle part of gels is shown as loading control (–). The upper right panel shows a time-course analysis of the VIC-oligo 4 effect on translation. The lower panel shows a polysome profile of MEFs transfected with VIC-oligo 4 (red) or with unconjugated oligo 4 (black). Extracts were separated in a 10–40% sucrose gradient and fractionated as described in the Materials and methods; the identities of the main peaks are indicated. (c) Fusion protein-mediated blockage of ES6S affects global translation. Model of EGFP (green) fused to eS4 (orange) of the 40S subunit. Note that in this model of human 40S, the ES6SA helix is in an ‘outward’ orientation (upper left panel). Fluorescence microscopy of HeLa cells expressing eS4–EGFP fusion protein (upper right panel). Micrographs were taken at 36 h. Nuclei are encircled by a dashed line; note the bright nucleolar and cytoplasmic staining. The bottom panel shows the distribution of eS4 and eS4-EGFP proteins in a 10–35% sucrose gradient from HEK293T cells transfected with eS4-EGFP. (d) Measurement of total protein synthesis by OPP fluorescence in transfected cells expressing no/low (black line) or high levels of eS4-EGFP (green line). Cells were first gated into two groups according to EGFP expression, and then the distribution of OPP fluorescence intensity was determined (left panel). The right panel shows OPP fluorescence measurements for cells transfected with eS4 alone, cells treated with 50 μg/ml CHX for 20 min, and cells transfected with eS4–EGFP that express (or do not express) EGFP. Data are the mean of three independent experiments ± standard deviations (SD).