Fig. 3: Regulation of global protein expression by NEDD8 in human triple-negative breast cancer (TNBC) cells.

Label-free protein quantification was performed using mass spectrometry in the MDA-MB-231 wild-type (WT)/NEDD8 knock-out (KO) cell line pair using 4 replicate samples of each line. a Up-regulated (red) and down-regulated (blue) proteins upon NEDD8 deletion were shown in a volcano plot. A Welch’s unequal variances T-test was applied to determine differences in protein expression between control and KO cells. The False Discovery Rate was calculated to adjust the p values. b Differentially expressed proteins and unique proteins were divided into either upregulated in NEDD8 KO or upregulated in control cells for pathway analysis. A hypergeometric test was conducted to determine enriched pathways from the Reactome and Gene Ontology Biological Process collections. p values were adjusted with Benjamini–Hochberg correction. c Interaction of changed proteins in the WT/KO cell line pair was grouped based on biological processes. Purple: unique in WT, Brown: unique in KO. Color is based on Log2 fold changes between KO and WT cells. d Expression of UBE2T was measured by Western Blotting. Representative image of 3 independent repeats was shown. e Control or KO MDA-MB-231 cells were treated with a UBA1 inhibitor, TAK-243, at 1000, 400, 100, 10 nM or 0.1% DMSO. Cell proliferation was measured by live-cell imaging. Representative experiment of 2 independent repeats. Control or KO MDA-MB-231 cells were treated with (f) pevonedistat or (g) TAK-243 at 1000, 400, 100, 10 nM or 0.1% DMSO. Cells were harvested at 24 h and the expression of CDT1 was measured using western blotting. Representative western blot of 2 independent repeats. Source data are provided as a source data file.