Figure 3.

Generation of p21-degron lines. (a) Western blot of whole-cell extract from indicated cell lines probing for p21, indicating all p21 expressed in the hTert-RPE1 OsTIR1 mRuby-PCNA p21-mVenus-mAID-SMASh cell line is tagged with mVenus-mAID. (i) Cells were reverse transfected with the indicated siRNAs, NTC or p27 and collected after 48 h; 10 µM etoposide (ETO) was added 24 h prior to sample collection to induce DNA damage as p21 expression is low in untreated hTert-RPE1 cells. p21-mVenus-mAID has a predicted molecular weight of 55 kDa; no p21-mVenus-mAID-SMASh is detected as the SMASh tag self-cleaves from the protein. Vinculin was used as a loading control. (ii) Western blot of whole-cell extract indicating that p21-mVenus-AID-SMASh is degraded following DIA (doxycycline, IAA and ASV) addition after induction of p21 expression by ETO. ETO and DIA: doxycycline (1 µg ml⁻¹), IAA (500 µM) and ASV (3 µM) were added 24 h before sample collection. (b) hTert-RPE1 mRuby-PCNA p21-Venus-AID-SMASh cells were reverse transfected with the indicated siRNAs, 6 h later DMSO or DIA were added as indicated and 24 h DMSO or palbociclib added for 48 h. Cells were pulse labelled with EdU before fixation and (i) EdU incorporation and (ii) G1 percentage were quantified. Data from n = 3 repeats plotted as SuperPlots. One-way ANOVA with Sidak's multiple comparisons test was used to compare palbociclib-treated samples with control (siNTC DMSO palbociclib), all differences were non-significant.