Fig. 2: IBG1-induced degradation of BRD2 and BRD4 is dependent on CRL4–DCAF16.

a, Schematic of FACS-based CRISPR–Cas9 screens. Doxycycline (Dox)-inducible Cas9 (iCas9) KBM7 BRD4–BFP reporter cells were transduced with a CRL-focused sgRNA library, treated with BET degraders and sorted based on BRD4–BFP/mCherry ratios. b, FACS-based CRISPR screens for BRD4 stability. KBM7 iCas9 BRD4 reporter cells were treated with DMSO, MZ1 (10 nM) or IBG1 (1 nM) for 6 h before sorting. 20S proteasome subunits, COP9 signalosome subunits and E1 and E2 ubiquitin enzymes inside the scoring window (one-sided MAGeCK P value < 0.01, fold change > 1.5) are highlighted. c, CRISPR–Cas9 viability screen. HCT-116 cells were transduced with Cas9 and a ubiquitin–proteasome system-focused sgRNA library and treated with IBG1 (58 nM; fourfold half-maximal inhibitory concentration (IC₅₀)) for 6 days. Genes with a fold change > 2 and one-sided MAGeCK P value < 0.01 are highlighted. d, Screen validation. KBM7 iCas9 BRD4–BFP reporter cells were transduced with AAVS1, DCAF16 or DDB1-targeting sgRNAs, treated with DMSO, IBG1 (1 nM) or dBET6 (10 nM) for 6 h, and BRD4–BFP was quantified by FACS. e, DCAF16 knockout and rescue. KBM7 iCas9 BRD4–BFP reporter cells were transduced with AAVS1 or DCAF16-targeting sgRNAs, with or without sgRNA-resistant DCAF16 cDNA. After knockout of endogenous DCAF16, cells were treated for 6 h as in b and BRD4–BFP was quantified by FACS. f, Apoptosis induction. Wild-type or DCAF16-knockout KBM7 cells were treated with indicated concentrations of dBET6 or IBG1 for 16 h. Cleaved PARP1 was evaluated by immunoblotting. g, Viability assay. Wild-type or DCAF16-knockout KBM7 cells were treated with IBG1 or dBET6 for 72 h and cell viability was evaluated by CellTiterGlo assay. Mean ± s.d. of n = 3 biological replicates. h, Fluorescence polarization binary binding assay. FITC-labelled sulfonamide probe (Supplementary Methods) was titrated into DCAF15–DDB1(ΔBPB)–DDA1 or DCAF16–DDB1(ΔBPB)–DDA1. DDB1(ΔBPB) lacks the cullin-binding ___domain (BPB). n = 3 technical replicates. d–f, n = 3 independent experiments. d–h, Mean ± s.d.