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. 2023 Jul 27;14:4527. doi: 10.1038/s41467-023-40132-0

Fig. 3. SA-β-Gal staining marks long durations of cell-cycle withdrawal and is correlated with increased lysosomal content and autophagic flux.

Fig. 3

a Schematic of the CDK2 activity sensor. The sensor localizes to the nucleus when unphosphorylated; progressive phosphorylation by CDK2 leads to translocation of the sensor to the cytoplasm. NLS, nuclear localization signal; NES, nuclear export signal; S, CDK consensus phosphorylation sites on serine. b MCF10A cells expressing the CDK2 activity sensor were treated with 10 μM etoposide for 24 h, washed, and subjected to time-lapse microscopy of CDK2 activity 6 d later for 96 h (from 6-10 d). The cells were fixed and stained for SA-β-Gal after the last frame was taken. c Left: Single-cell traces were clustered based on the top, middle, and bottom 10% of SA-β-Gal signal and the total hours CDK2low (below a cutoff of 0.8) was plotted for each bin. c Right: Untreated MCF10A cells were sorted by flow cytometry for the bottom 1% of mCitrine-Ki67 signal, plated and allowed to grow for 48 h, filmed for 48 h to monitor CDK2 activity, fixed and stained for SA-β-Gal, and were manually classified as SA-β-Gal positive versus negative. d, e Median SA-β-Gal signal for pRblow MCF10A cells pushed into quiescence by contact inhibition, serum starvation, 3 μM Palbociclib treatment, or 100 nM Trametinib treatment for 3-12 d, and fixed and stained for SA-β-Gal and phospho-Rb. Best fit lines were computed for each condition from the average of 6 technical replicates. Dots represent raw data points and open circles represent the mean of these data points. Percentages listed represent the proportion of cells that re-entered the cell cycle during a 5 d release from 2 weeks of treatment (see Supplementary Fig. 4c). f Model for SA-β-Gal accumulation as a function of cell-cycle exit time. g MCF10A cells were treated with 10 μM etoposide for 24 h, washed, fixed after 3 d, and stained for SA-β-Gal and LAMP1. h, i Same experimental scheme as described in g. Cells were fixed and stained for SA-β-Gal, LC3II, and LAMP1 after a 3 h treatment with 50 μM chloroquine.