Fig. 2 |. AMBRA1 targets D-type cyclins for ubiquitin-mediated degradation, controlling cell cycle progression.

a, Three AMBRA1^−/− (KO) and two AMBRA1^+/+ HCT-116 clones were treated with cycloheximide (CHX) as indicated and lysates were blotted with the indicated antibodies. b, HCT-116 cells expressing 2×Flag–mAID–AMBRA1 (clone F1) were treated with DMSO or doxycycline (Dox) for 12 h before exposure to auxin (Aux). HA, haemagglutinin. c, HCT-116 cells expressing 2×Flag–mAID–AMBRA1 were treated as in b, washed, and cultured in growth medium as indicated. d, HCT-116 cells expressing 2×Flag–mAID–AMBRA1 were pre-treated with DMSO or doxycycline for 12 h, and exposed to combinations of auxin, doxycycline, MG132 or MLN4924 for 4 h. Endogenous cyclin D1 was immunoprecipitated in denaturing conditions and probed with ubiquitin (Ub) and cyclin D1 antibodies. e, HCT-116 cells were transfected with siRNAs as indicated (E2F1/E2F2/E2F3, E2F1, E2F2 and E2F3 siRNA; Extended Data Fig. 5h) before pulsing with EdU for 45 min. Cell cycle profiles were measured by flow cytometry. Data are mean ± s.e.m. (n = 4 biological replicates per group). Adjusted P values were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant. f, HCT-116 cells were transfected with a non-targeting (NT) siRNA or an siRNA against AMBRA1 before treatment with palbociclib or abemaciclib for 24 h. g, Ratio of cytoplasmic to nuclear CDK4/6 activity was measured in individual cells (Extended Data Fig. 5i). Violin plots show median (dashed line) and quartiles (dotted lines). P values were calculated using the non-parametric Mann–Whitney, unpaired t-test between AMBRA1 WT (n = 642 cells) and AMBRA1 KO (n = 738 cells). Unless otherwise noted, experiments were performed at least three independent times. Asterisks indicate non-specific bands.