Fig. 2: Rescue of cell proliferation by alternative mechanisms in NEDD8 deficient cells.

The NEDD8 gene was deleted using CRISPR/Cas9 in three human triple-negative breast cancer (TNBC) cell lines, i.e., MDA-MB-231, HCC1937 and BT549. a Expression of the NEDD8 protein was measured using Western Blotting (representative blot of three independent experiments was shown) and (b) cell proliferation of the wild-type control (WT ctrl) and NEDD8 knock-out (KO) cells was quantified in a live-cell imaging system. Representative experiment of three independent experiments were shown. c Genome-wide CRISPR screens were performed in MDA-MB-231 WT or NEDD8 KO cells and the gRNA frequencies were compared between day 21 and day 4 (depleted genes in blue and enriched genes in purple). Data were processed in the MAGeCK pipeline and p values were calculated from the negative binomial model. Log2 fold changes were plotted against the Log10 p values in volcano plots with highlighted gene hits. One genome-wide CRISPR screen was performed. d MDA-MB-231 WT or NEDD8 KO cells were treated with the glutathione peroxidase 4 (GPX4) inhibitor (ML210), and the dose-dependent effects on cell proliferation were quantified using a live-cell imaging system. Representative experiment of three independent replicates. e Potency of a (NEDD8-activating enzyme) NAE inhibitor, pevonedistat, on the WT or NEDD8 KO TNBC cell lines was shown at 84 h. Representative experiment of three independent replicates. f Number of uniquely or commonly depleted genes in the WT MDA-MB-231 or NEDD8 KO cells was shown in a Venn diagram. g Pathway analysis on uniquely depleted genes in MDA-MB-231 WT or NEDD8 KO cells in the genome-wide CRISPR screens. Enrichment analysis was conducted using hypergeometric test and Benjamini–Hochberg adjusted p values are reported. h Illustration of the conditional essentiality model of the NEDD8 gene in TNBC cells. Source data are provided as a source data file.