Extended Data Fig. 7 |. AMBRA1 binding to CUL4 is required for regulating cyclin D.

a, Co-immunoprecipitation of transfected AMBRA1–Myc–Flag and cyclin D–HA (D1, D2 or D3) in 293T cells, analysed by immunoassay. b, Co-immunoprecipitation of transfected Myc-tagged cullin proteins with endogenous AMBRA1 in U2OS cells, analysed by immunoassay. c, RT–qPCR analysis of CCND1 mRNA expression in U2OS cells following knockdown of AMBRA1 or various cullin genes by siRNA pools (n = 3 biological replicates). d, Co-immunoprecipitation of transfected wild-type (WT) AMBRA1 and AMBRA1(ΔH) with endogenous CUL4A and CUL4B in 293T cells. e, Immunoassay of AMBRA1 in control and AMBRA1-knockout U2OS cells with doxycycline-inducible expression of wild-type AMBRA1, AMBRA1(ΔH) or GFP control, after treatment with 500 ng ml⁻¹ doxycycline (+Dox) or DMSO (−Dox) for 2 d. f, Immunoassay of cyclin D1 ubiquitylation in 293T cells with overexpression of wild-type AMBRA1 or AMBRA1(ΔH). Cells were pretreated with 1 μM bortezomib for 3 h and lysed in denaturing conditions before immunoprecipitation of cyclin D1. Representative of two independent experiments. g, Immunoassay of cyclin D1–HA in U2OS cells expressing wild-type cyclin D1 or phosphomutant cyclin D1 (cyclin D1(T286A)) treated with 10 μg ml⁻¹ cycloheximide for up to 2 h. Cells were transfected with control or AMBRA1-targeted siRNA pools 3 d previously. h, Quantification of cyclin D1–HA protein levels in U2OS cells from g with best-fit curves for one-phase decay (n = 3 biological replicates). i, Co-immunoprecipitation of cyclin D1–HA (wild-type or T286A) and endogenous AMBRA1 in U2OS cells. CDK4 serves as a positive control for cyclin D1 binding. Representative of two independent experiments. All data are mean ± s.d. P values calculated by two-sided paired t-test (c) and two-way ANOVA (h). HSP90 and actin are loading controls.