Extended Data Fig. 11 |. Loss of AMBRA1 promotes the formation of CDK2–D-type cyclins complexes.

a, Schematic representation of the experiment used to identify global regulators of the response to CDK4/6 inhibitors in U-2 OS cells using a whole-genome, CRISPR–Cas9 human GECKOv2 pooled library. b, Scatter plot of the candidate hits generated from a. This experiment was performed once, with four technical replicates. P values were calculated using the MAGeCK algorithm¹⁴. c, Parental and AMBRA1^−/− HCT-116, T98G or U-2 OS cells were treated with various concentrations of abemaciclib. Survival curves were generated using the AlamarBlue assay. Data are mean survival percentage ± s.d. (n = 3 biological replicates per group). The signal from each condition was normalized to an untreated control to adjust for any relative growth difference. d, AMBRA1^+/+ and AMBRA1^−/− T98G cells were treated with abemaciclib (75 nM) or DMSO for 24 h before collection and immunoblotting for the indicated proteins. e, HCT-116 cells were transfected with a non-targeting siRNA or an siRNA against AMBRA1 for three rounds before treatment with abemaciclib (75 nM) or DMSO for 24 h. Cells were collected and their extracts were immunoblotted for the indicated proteins. f, Parental and AMBRA1^−/− HCT-116 cells were collected, and their extracts were used for an immunoprecipitation with an anti-CDK4 antibody or control IgG isotype. Proteins were immunoblotted as indicated. g, HEK 293T were transfected with plasmids expressing either FFSS-tagged cyclin D1, cyclin D2 and cyclin D3 or empty vector before lysis and immunoprecipitation with an anti-Flag resin. Proteins were immunoblotted as indicated. h, AMBRA1^−/− HCT-116 cells were collected, and their extracts were used for an immunoprecipitation with an anti-cyclin D1 antibody or control IgG isotype. Proteins were immunoblotted as indicated. i, AMBRA1^−/− HCT-116 cells were collected, and their extracts were used for an immunoprecipitation with an anti-cyclin D3 antibody or control IgG isotype. Proteins were immunoblotted as indicated. j, HCT-116 cells were infected with retroviruses expressing either FH-tagged cyclin D1(WT) or cyclin D1(T286A), and their extracts were used for immunoprecipitation with anti-CDK2 antibody or control IgG isotype. Proteins were immunoblotted as indicated. Unless otherwise noted, experiments were performed at least three independent times.