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. 2021 Nov 17;11(11):210125. doi: 10.1098/rsob.210125

Figure 2.

Figure 2.

p21 and p27 are not required for palbociclib-mediated arrest in hTert-RPE1 cells. (a) hTert-RPE1 WT and p21KO cells were reverse transfected with siNTC or sip27 and treated with DMSO or palbociclib 6 h after plating, as indicated. Cells were pulse labelled with 10 μM EdU for 30 min before fixation 48 h following drug treatment. EdU-positive cells quantified as cells with a nuclear : cytoplasmic ratio of EdU signal as greater than or equal to 1.2. (i) Representative images. Scale bar, 100 μm. (ii) Cells were classified in G1 phase according to their DNA content as quantified by Hoechst nuclear sum intensity. (iii) EdU positive cells quantified as cells with a nuclear : cytoplasmic ratio of EdU signal as greater than or equal to 1.2. Data plotted as a SuperPlot [55] and in subsequent figures: dots represent replicate wells, colour coded by experimental repeat, triangles represent mean values for each of n = 3 experimental repeats with mean and s.e.m. shown. One-way ANOVA with Sidak's multiple comparisons test was performed between mean values of palbociclib treated samples in comparison to WT siNTC palbociclib, all differences were non-significant. (b) hTert-RPE1 mRuby-PCNA p21 WT and p21KO cells were transfected with siNTC or sip27 and 6 h later treated with the indicated concentrations of palbociclib for 48 h before 30 min EdU pulse and fixation. Plot of percentage of EdU-positive cells, (i), and of cells in G1 phase, (ii). Mean and s.d. from one of n = 2 experimental repeats shown. One-way ANOVA with Sidak's multiple comparisons test was performed between WT and p21KO at each concentration of palbociclib; all differences were non-significant. (c) Graphs show timing of cell cycle arrest when palbociclib is added to asynchronous WT and p21KO cells (at 0 min) 6 h after transfection with the indicated siRNAs. Cell cycle phenotypes were monitored by live-cell imaging following drug addition and tracked manually over 24 h using mRuby-PCNA as a readout. Three fields of view were quantified per condition. Each row represents an individual cell. (i) WT NTC siRNA n = 68, (ii) WT p27 siRNA n = 61, (iii) p21KO NTC siRNA n = 73 and (iv) p21KO p27 siRNA n = 70 cells.