Extended Data Fig. 5 |. Cyclin D mediates the phenotypes of AMBRA1- mutant cells.
a, Immunoassay of cyclin D1, D2, and D3 in wild-type U2OS cells overexpressing all three D-type cyclins from the same lentiviral vector or RFP as a control. b, Representative flow cytometry plots of BrdU and PI staining in cells from a treated with increasing doses of palbociclib for 24 h. c, Percentage of cycling S-phase cells from b (n = 3 biological replicates). Data are mean ± s.d. P values calculated by two-way ANOVA (Pcell line < 0.0001) with post hoc Sidak test. d, Representative flow cytometry plots of BrdU and PI staining in U2OS cells overexpressing stabilized cyclin D1(T286A)–HA or RFP control, treated with increasing doses of palbociclib for 24 h. e, Representative flow cytometry plots of BrdU and PI staining in control and AMBRA1-knockout U2OS clones after 48 h of cyclin D1 (CCND1) knockdown with siRNA pools. f, Co-immunoprecipitation of p27 in control, knockout and cyclin-D1(T286A)-overexpressing U2OS cells, and immunoassay of relevant protein complexes (n = 2 biological replicates). HSP90 is a loading control.