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. Author manuscript; available in PMC: 2021 Jan 12.
Published in final edited form as: Cell Rep. 2020 Jul 14;32(2):107901. doi: 10.1016/j.celrep.2020.107901

Figure 1. Measuring the Duration of Cell Cycle Phases Using Fluorescently Labeled PCNA and Histone H2B in MCF10A Cells.

Figure 1.

(A) Schematic of the regulation of Cdk1 activity at the G2/M transition by cyclins and multiple feedback loops. The protein synthesis inhibitor cycloheximide (CHX) can block cyclin accumulation; it also activates p38 MAPK, which can delay G2/M progression by inhibiting Cdc25 and/or potentially activating Wee1/Myt1 (Reinhardt and Yaffe, 2009). The small-molecule inhibitors SB202190 and SB203580 and PD0166285 and MK-1775 have been used in this study to inhibit p38 MAPK or Wee1/Myt1 activity, respectively.

(B) eYFP-PCNA can be used to determine the onset of S phase, the completion of S phase, and the onset of mitosis (nuclear envelope breakdown); histone H2B-mTurquoise (used here) or histone H2B-mCherry can be used to determine anaphase onset. Scale bars: 10 μm.

(C) Three examples of cells showing the disappearance of eYFP-PCNA foci (yellow arrows) at the end of S phase. Times (in the format h:min) were aligned to the time of entry into G2 phase. Scale bars: 10 μm.

(D) Frequency distributions of G2 phase duration measured in MCF10A cells expressing H2B-mCherry and eYFP-PCNA either in the absence (“medium,” gray; n = 104) or presence of 0.1% DMSO (blue; n = 100). Means and standard deviations are indicated.