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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 5. Cycloheximide Treatment in S Phase Blocks Cell Cycle Progression, Even in the Absence of Wee1/Myt1 Activity.

Figure 5.

(A) Cell cycle progression for MCF10A cells expressing H2B-mCherry and eYFP-PCNA treated during S phase with either DMSO (n = 99), cycloheximide (n = 100), 1 μM MK-1775 (n = 100), cycloheximide plus 1 μM MK-1775 (n = 100), or 4 μM MK-1775 alone (n = 99). Each row represents a single cell. Rows marked with a purple square denote cells that underwent abnormal mitoses, often lacking proper metaphase and cytokinesis.

(B) Cell cycle progression for MCF10A cells expressing H2B-mCherry and eYFP-PCNA treated during S phase with either DMSO (n = 99), cycloheximide (n = 98), 1 μM PD0166285 (n = 99), or cycloheximide plus 1 μM PD0166285 (n = 105). Each row represents timing data from a single cell. Rows marked with a purple square denote cells that underwent abnormal mitoses, often lacking proper metaphase and cytokinesis.

(C) Montage of an MCF10A cell expressing H2B-mCherry and eYFP-PCNA treated with cycloheximide during S phase. In this cell (and most cells), the PCNA foci became weaker after cycloheximide treatment, yet they never completely disappeared, suggesting that these cells remained in S phase. Time (in the format h:min) was aligned to the point of cycloheximide addition.

(D) Montage of an MCF10A cell expressing H2B-mCherry and eYFP-PCNA treated with 1 μM PD0166285 during S phase that progressed into mitosis in the presence of PCNA foci (suggesting that the cell never completed S phase). Time (in the format h:min) was aligned to the point of drug addition. Note the abnormal mitotic progression without a proper metaphase and cytokinesis.

(E) Mitotic duration, measured as the time from nuclear envelope breakdown (NEB) to anaphase, for cells treated either in G2 phase or S phase with DMSO, PD0166285, or cycloheximide plus PD0166285. Whereas treatment with PD0166285 or treatment with PD0166285 plus cycloheximide in G2 phase only slightly extended mitosis (see also Figure 7), treatment with these drugs in S phase dramatically increased the duration of mitosis.