Figure 4.
Palbociclib-dependent arrest can be maintained in the absence of p21/p27. (a) hTert-RPE1 p21-degron cells were treated with DMSO/palbociclib, transfected with the indicated siRNAs 18 h later and then treated with DIA 6 h following transfection. Cells were pulse labelled for 30 min with 10 µM EdU and fixed 48 h following DIA addition. (b) hTert-RPE1 WT and p21KO cells were treated with DMSO/palbociclib and transfected with the indicated siRNAs 18 h after drug treatment; 48 h after transfection addition, cells were pulse labelled with 10 µM EdU for 30 min and fixed. (c) hTert-RPE1 mRuby-PCNA p21GFP cells were treated with DMSO for 72 h or palbociclib for the indicated times, and p21GFP levels quantified. (i) Representative frequency distribution of measured intensities from one experimental repeat shown, p21High threshold for this experiment shown as a dotted line. (ii), Percentage of cells classified as p21 high above a threshold of p21 intensity, n = 3 experiments shown. Data were normalized to 72 h DMSO treatment within each experimental repeat. (d) hTert-RPE1 p21-degron cells were treated with DMSO/Palbocilib for 72 h, transfected and then DMSO/DIA added 6 h later. Cells were pulse labelled with 10 µM EdU for 30 min 48 h after DIA addition. (a,b,d) Data from n = 3 repeats plotted as SuperPlots. One-way ANOVA with Sidak's multiple comparisons tests were used to compare palbociclib-treated samples, all differences were non-significant.