UBE2D3 and UBE2G1 cooperate to polyubiquitinate lenalidomide-induced substrates of CRL4^CRBN. (A) HEK293T single-cell clones in which UBE2G1 and/or UBE2D3 were knocked out via CRISPR-Cas9 introduction of frameshifting DNA edits were transduced with an the IKZF3 degron reporter and then treated with a titration of lenalidomide. After 20 hours, the EGFP/mCherry ratio was assayed via flow cytometry. Data points are an average of 3 experimental replicates and error bars represent standard error of the mean. Corresponding EC50 values are shown for each genotype, with error bars representing the 95% confidence interval. (B) An in vitro ubiquitination reaction containing recombinant CRL4^CRBN, and HA-IKZF3 (aa146-168) derived from HEK293T cells, and the indicated reaction components was run for 30 minutes and immunoblotted (IB) for HA. Data are representative of 3 experimental replicates. (C) In vitro CRBN autoubiquitination reactions in which UBE2D3 or UBE2G1 were added sequentially, with each enzyme individually incubated with CRBN for 30 minutes followed by addition of the second E2 enzyme for the indicated amount of time. In vitro ubiquitination reactions were immunoblotted as indicated. (D) MM1.S-Cas9 cells were infected with gRNAs targeting the indicated genes, treated with DMSO or 1 μM lenalidomide for 20 hours, then lysates were harvested and immunoblotted as indicated. Data are representative of 3 experimental replicates. (E) NCIH929-Cas9 cells were infected with gRNAs targeting the indicated genes, treated with DMSO or 0.1 μM pomalidomide for 20 hours, then lysates were harvested and immunoblotted as indicated. (F) NCIH929-Cas9 cells infected with control or UBE2G1 gRNAs were treated with DMSO, pomalidomide, or CC-122 for 10 days, with dosing at day 0 and day 5. Dose response curves show viability normalized to DMSO treatment. Data points are an average of 3 experimental replicates and error bars represent standard error of the mean for each drug concentration.