Figure 5.

Detection of the translational response of an exogenous reporter mRNA in 1,000 human cells with R-MIPs. (a) The absorbance profile at 254 nm across a sucrose gradient is shown at the top, and the positions of the 40S and 60S ribosomal subunits, 80S monosomes, and polysomes are indicated. The black line represents a profile of co-transfected HeLa cells grown in serum-starved conditions (starved); the red line upon stimulation of the cells with serum (refed). The bar chart depicts the distribution of RPS6-GFP (left) and Luc control (right) mRNAs in subpolysomes (fractions 1–4) and polysomes (fractions 5–12) of starved and refed cells. RT-PCR data was normalised to a spiked-control RNA (LysA) added to each fraction prior to RNA isolation to adjust for technical variation during RNA isolation. (b) Bar chart depicting the changes of RPS6-GFP relative to Luc mRNA in refed (R) vs. starved (S) conditions in MIP/NIP eluates and in polysomes. RT-qPCR data was normalised to the respective mRNA levels in cell extracts (input) to adjust for variation in the transcriptome upon treatment of cells (see Methods). The change in polysomes was analysed upon pooling fractions 5–12 from the sucrose gradient shown in a. Error bars represent SEM, n = 3. **P < 0.01, two-tailed homoscedastic student’s t-test.