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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Nature. 2021 Apr 14;592(7856):794–798. doi: 10.1038/s41586-021-03474-7

Fig. 1 |. AMBRA1 loss regulates the response to CDK4/6 inhibition as well as levels of cyclin D.

Fig. 1 |

a, Volcano plot of a CRISPR–Cas9 screen for genes that regulate the response to palbociclib in U937 cells, analysed using the Cas9 high-throughput maximum likelihood estimator (casTLE). FDR, false-discovery rate. b, Immunoassay for AMBRA1 or RB in control and AMBRA1- or RB1-knockout U937 cl ones. sgAMBRA1 no. 1 and no. 2 denote two different sgRNAs against AMBRA1; sgCtrl, control sgRNA. c, Change in U937 cell numbers after a 48-h treatment with 0.5 μM palbociclib or DMSO. d, BrdU and propidium iodide staining analysis of cycling S-phase U937 cells treated with 0.5 μM palbociclib for 24 h. Each symbol in c, d is an isogenic clone (n = 3 biological replicates per clone). e, Immunoassay of RB phosphorylation (at S795) in U937 cells treated with increasing doses of palbociclib or DMSO (−) for 24 h. f, Immunoassay of G1 cyclins and cyclin-dependent kinases in U937 clones. U937 cells do not express cyclin D1. g, Volcano plot of shotgun mass spectrometry comparing control and AMBRA1-knockout (KO) U2OS cells. Significant hits (|log2-transformed fold change| > 1, adjusted P < 0.05) are in red. BH, Benjamin–Hochberg. h, Immunoassay of cyclin D1 and haemagglutinin (HA) in U2OS cells overexpressing HA-tagged, stabilized cyclin D1 (cyclin D1(T286A)–HA) or red fluorescent protein (RFP) control. i, Analysis of cycling S-phase cells from h treated with increasing doses of palbociclib for 24 h (n = 3 biological replicates). j, Top, analysis of cycling S-phase U2OS cells after cyclin D1 (CCND1) knockdown by siRNA pools. Bottom, corresponding immunoassay 48 h after siRNA transfection (n = 3 biological replicates). NT, non-targeting control. k, l, Co-immunoprecipitation (IP) of cyclin D1 (k) and CDK2 (l) in control, AMBRA1-knockout and cyclin-D1(T286A)-overexpressing U2OS cells, and immunoassay of relevant protein complexes (n = 1 (k) or n = 2 (l) biological replicates). Tubulin and HSP90 are loading controls. All data are mean ± s.d. P values calculated by two-sided unpaired t-test (c, d), negative binomial test (g), two-way analysis of variance (ANOVA) with post hoc Sidak test (i, ANOVA Pcell line < 0.0001) and two-sided paired t-test (j).