Fig. 2. Quantifying SA-β-Gal in single cells reveals a graded signal with overlap between treated and untreated cells and the presence of SA-β-Gal^pos cycling cells.

a A representative single MCF10A cell stained for SA-β-Gal and imaged in pseudo-color-brightfield at 6 d after release from a 24 h treatment with 10 μM etoposide. An intensity profile (dotted line) was taken from each channel of the RGB stack. We define the SA-β-Gal signal as the 5^th percentile of the red pixels in the cytoplasm of each cell. b Distribution of SA-β-Gal signal in untreated cells or in cells released for 6 days after release from a 24 h treatment with 10 μM etoposide, with four representative single cells at increasing intensities of staining. c Heterogeneity in co-staining of SA-β-Gal and Rb phosphorylation by immunofluorescence in MCF10A cells released for 6 d from a 24 h treatment with 10 μM etoposide. Percentages reflect the fraction of cells with each behavior using the cutoffs in d. d Scatter plots of SA-β-Gal versus phospho-Rb from the same cells in c. Heatmap represents the density of data points. e The same data from Fig. 1d for etoposide-released cells that entered the cell cycle during live-cell imaging. Cells were split into those that completed a cell cycle during the 4 days of imaging but were Ki67^off again by the final frame of the movie (N.C. at End) vs. those that were in the cell cycle and Ki67^high on the final frame of the movie (Cyc. at End). Cells were fixed and stained for SA-β-Gal after the last frame of the movie was taken and each cell’s SA-β-Gal signal was linked to its Ki67 history.