Asynchronously cycling MCF10A cells as measured by flow cytometry. Thirty-three percent of mitotic (pHH3+) cells

Asynchronously cycling MCF10A cells as measured by flow cytometry. Thirty-three percent of mitotic (pHH3+) cells have high p21 levels; 12 of mitotic cells have falling pRb, but there is no Thonzylamine Epigenetics discrete hypophosphorylated Rb population (n = ten,000 cells). Gates have been set as outlined by the saddle point for p21 and pRb. (C) Asynchronously developing MCF10A cells were stained for pHH3, pRb (S807/811), and Hoechst. Mitotic cells have been identified based on Hoechst staining and displayed as mock film strips. Anaphase cells are ordered based on distance in between chromosomes. Histone H3 is dephosphorylated starting in late anaphase (green stars), whereas Rb just isn’t dephosphorylated until immediately after anaphase is total and chromatin decondensation begins. (D) pRb (S807/811) in MCF10A cells from Fig. 2B. Shown are newly born cells fixed 12 min (Upper) or 24 min (Lower) right after anaphase was detected. Cells are ordered according to chromatin decondensation and distance between sister cells. The numbers of cells with hyper- and hypophosphorylated Rb presented here are proportional towards the total population. Green stars mark cells which are pHH3-; magenta stars mark cells with hypophosphorylated Rb.previous literature (270), whereas Rb will not be dephosphorylated till following late anaphase (Fig. three C and D), explaining why no discrete pHH3+/hypophosphorylated Rb population is detectable by flow cytometry. Synthesizing our flow cytometry, film strip, and time-lapse microscopy information (where we’ve got immunofluorescence data for phospho-Rb starting 12 min just after anaphase), we conclude that Rb dephosphorylation in CDK2low cells occurs in telophase following chromosome decondensation has started. The differential timing of p21 up-regulation in G2 and Rb dephosphorylation in telophase suggests that p21 up-regulation (by inhibiting CDK activity) causes entry into the CDK2low state,Moser et al.whereas Rb dephosphorylation (as a result of mitotic phosphatase activity as well as a dearth of CDK activity as CDK2low cells exit mitosis) is often a consequence.p21 Up-Regulation Precedes Mitosis in Mother Cells Whose Daughters Enter the CDK2low State Following Mitosis. The similarity betweenthe fraction of cells exiting the cell cycle immediately after anaphase plus the fraction of mitotic cells with higher p21 prompted us to ask when specifically the up-regulation of p21 occurs. Given the value of p21 in controlling the bifurcation in CDK2 activity (14, 20, 21, 31), we applied CRISPR-Cas9 to engineer MCF10A that express mCitrine-tagged p21 in the endogenous CDKN1A locus (SI Appendix, Figs. S3 and S4). Although some studies have highlighted the possible for N-terminal acylation or ubiquitylation as a regulatory mechanism for p21 (32, 33), we targeted the N terminus of p21 for the reason that similarly tagged constructs have been shown to keep functional p21 (14, 31). p21 tagged at the N terminus localizes ordinarily, degrades similarly to wild-type p21, can nevertheless interact with CDK2, and does not drastically alter cell cycle dynamics (SI Appendix, Fig. S4). We also examined three other cell lines in which p21 is tagged at the C terminus having a fluorescent protein at the endogenous locus [RPE-hTERT (21), MCF7, and HCT116 (34)] or expressed from an inducible promoter at endogenous levels [U2OS (35)]. Each cell line was transduced with a fluorescently tagged H2B nuclear marker plus the CDK2 sensor and then imaged for 24 h (Movies S2 6). Cells from every population had been classified based on CDK2 activity just after anaphase: CDK2inc , CDK2low , and cells that.