Fig. 5: Protein biosynthesis declines along the quiescence-senescence continuum.
A MCF10A cells were treated with 2.5 μM, 10 μM, or 25 μM etoposide for 24 h, followed by a 6 d drug-free recovery. Cells were treated with OPP for 30 min prior to fixation. Each cell’s OPP intensity was normalized to the cell area and plotted as histograms for each etoposide dose. Number of cells and replicates in Supplementary Data 2. B Experimental timeline (top). MCF10A cells expressing the CDK2-activity reporter and a DHFR-mCherry translation reporter were imaged by live-cell microscopy for 96 h from 6d–10d after etoposide washout. TMP was added at the start of the gray shading to induce the DHFR-mCherry reporter, and the mCherry signal (each cell normalized to its average signal before TMP) was plotted for fast-cycling, slow-cycling, or predicted-senescent cells for each dose of etoposide. Dark line is the mean for each group, shading is the 95% confidence interval. Gray shading indicates the frames used to calculate the mCherry signal slope plotted in (C). Number of cells and replicates in Supplementary Data 2. C A line was fit to the initial slope of mCherry accumulation after TMP addition. The average slope for each cell-cycle behavior group for each dose is shown. Error bars represent 95% CI. PS is predicted-senescent. Number of cells and replicates in Supplementary Data 2. D All significantly downregulated pathways from the RNA-processing and translation pathway groups in Fig. 3C for senescent clusters 4 and 9 were broken down into more specific pathway groups. E Average scaled expression for select leading-edge genes by cluster from groups of pathways in (D). F Heatmap of all GO biological processes from the RNA-processing and translation pathway groups over pseudotime. Colormap represents the Loess fit value for each module score over pseudotime. Module scores were rescaled for visualization on the same scale. For full pathway names, see Supplementary Data 1. G Cells were treated with 2.5 μM, 10 μM, or 25 μM etoposide for 24 h followed by a 6 d drug-free recovery. OPP was multiplexed with RNA FISH for three SASP genes and imaged. RNA FISH puncta number and total OPP per cell were quantified and plotted as a single-cell density scatter. Red line is drawn at the 95th percentile of untreated cells. Number of cells and replicates in Supplementary Data 2.
