Effect of Bro40 and Bro40mut on gene expression of wild type H1299 non-small cell lung cancer cell line

We previously discovered WDR4 as a Cullin 4 ubiquitin ligase substrate adaptor, which associates with poor cancer prognosis. With the help of ubiquitylome analysis, we uncovered PTPN23 as a substrate of WDR4. While PTPN23 is a component of ESCRT complex that facilitates EGFR lysosomal trafficking and degradation, the high expression of WDR4 and low expression of PTPN23 in patient sample suggesting that WDR4-mediated PTPN23 ubiquitination might contribute to PTPN23 proteasomal degradation and lead to poor patient survival rate. To prevent PTPN23 from WDR4-mediated ubiquitination, we used fine mapping to figure out the binding site of these two proteins and identified a 40-amino acid region on PTPN23 (Bro1 domain) can bind with WDR4, we named this 40-amino acid region as Bro40. Structural docking of WDR4 and Bro1 showed that the N-term of Bro40 is the favorable contact site. Thus, 6 amino acids at the N-term of Bro40 were mutated into alanine, so called Bro40mut. Bro40 and Bro40mut were fused on a GFP-tag plasmid for functional test and animal experiment. The tumor suppressing function of Bro40 was confirm by migration/invasion assays and animal model. To understand how Bro40 regulates cancer progression at RNA level, we transfected Bro40 and Bro40mut into H1299 cells followed by RNA sequencing. H1299 cells were transfected with Bro40 and Bro40mut for 24 hours, respectively. Comparative gene expression profiling analysis of high throughput RNA-seq data was obtained from 3 independent biological repeats. Bro40mut was set as control.