Fig. 4: CST1 relieves GPX4 ubiquitination through deubiquitinase OTUB1.

A GSEA analysis of our previous transcriptome sequencing data and GEO data set (GSE66229) enriched the deubiquitination pathway. B Predicting the deubiquitinase DUBs that interact with GPX4 through the online database (BioGRID, IntAct) and Venn analysis with all known DUBs. Intersection proteins include OTUB1 and OTUD5. C Co-IP assay on HEK293T cells transfected with Flag-tagged OTUB1 and OTUD5 plasmids. D WB detection of Flag, HA, Myc tagged proteins after IP in cells co-transfected with Myc-tagged CST1 and Flag-tagged OTUB1. E Endogenous Co-IP of GPX4 in MKN45-shNC/MKN45-sh1-CST1/MKN45-sh2-CST1. WB detection of IP proteins; OTUB1 binding to GPX4 was significantly reduced. F HGC-27-Vector/HGC-27-CST1 cells were transiently transfected with OTUB1 siRNA. The ubiquitination assay showed that the level of ubiquitin bound by GPX4 increased with the decrease of OTUB1, and the ubiquitination level of GPX4 was more obvious in HGC27 cells overexpressing CST1. G HGC-27-Vector/HGC-27-CST1 cells were transiently transfected with Flag-OTUB1-Con/WT/D88A and HA-GPX4 plasmids, WB detection showed that the GPX4 protein in the OTUB1-WT group was more stable, while the GPX4 protein in the OTUB1-D88A group was reduced. H Predicted binding complex models of CST1, OTUB1 and GPX4 by using the Cluspro online protein docking tool. I Schematic diagram of the construction of full-length and truncated CST1 proteins. J HEK293T cells were transfected with CST1-FL/N/C-Myc plasmids and OTUB1-Flag, GPX4-HA plasmid, respectively. Myc protein was immunoprecipitated, WB showed that CST1-FL and CST1-N truncated protein can bind to OTUB1, while CST1-FL and CST1-C truncated proteins can bind to GPX4. The SDS-PAGE used for separating CST fragments was 15%. (GSEA Gene set enrichment analysis, Con control/empty plasmid, WT Wild-type plasmid, D88A the D88 site of OTUB1 is point mutated to A plasmid).