Figure 1.

Palbociclib induction synchrony of hTERT-RPE1 cells. (a) hTERT-RPE1 cells were grown to 1.5 × 10⁴ cells cm² in DMEM (+10% serum), trypsinized and plated into 10 cm dishes at 4.4 × 10³ cm⁻². Six hours later, 150 nM palbociclib was added to the culture. After 24 h, cells were washed twice with pre-warmed medium before the addition of pre-warmed medium that contained 330 nM nocodazole before incubation for a further 24 h. Samples (one 10 cm dish per data point) were stained for propidium iodide FACS analysis at the following time points: just before palbociclib addition (U, untreated), at the switch from palcociclib to nocodazole medium (P) and 24 h after this switch to nocodazole (P + N). The strength of the nocodazole-induced spindle checkpoint arrest was revealed by the addition of nocodazole to an asynchronous population (U + N) for 24 h. The bimodal peak in the upper panel (U) shows 2 N (G1, left) DNA and 4 N (G2/M, right) DNA content of an asynchronous, untreated population. This experiment was repeated six times. (b) Cell populations were treated in the same way as (a) with the palbociclib concentration changed to the indicated value and one sample being left untreated. The average frequency of 2 N cells from at least three biological repeats is plotted for the palbociclib (grey bars) and nocodazole (blue bars) arrest points. Error bars show the limits of 1 s.d.. (c,d) hTERT-RPE1 cells were grown to around 1.5 × 10⁴ cells cm⁻² in DMEM (+10% serum), trypsinized and plated into 10 cm dishes at 4.4 × 10³ cm⁻². Twelve hours later, 150 nM palbociclib was added to the culture, before three washes in pre-warmed DMEM and sampling one dish every hour to generate the propidium iodide FACS profiles in (c) from which the plots of 2 N content shown in (d) were derived. The numbers next to the plots in c indicate time (hours) since release with U indicating an untreated control population. The 13–25 h plots (red (c): open squares (d)) were taken simultaneously alongside the 0–12 h population (grey (c): filled circles (d)). The synchronization shown in (c,d) has been performed six times and always reveals comparable results; however, variations in the precise synchronization profiles in each experiment (as can be seen in subsequent figures in the manuscript) mean that it is not appropriate to merge them into a single dataset.