Fig 2: CST1 is up-regulated in primary and metastatic GC tissues and is associated with a poor prognosis.Heatmap mainly showing expression levels of 20 up-regulated and 20 down-regulated different expressed genes in primary GC (A) and metastatic GC (C) vs. adjacent normal tissues (>1.5-fold). Volcano plots with differentially expressed genes in primary GC (B) and metastatic GC (D) vs. adjacent normal tissues. E Venn diagram representation of 768 overlapped up-regulated genes. F Histogram was shown the overlapped highest 20 up-regulated genes in these two clusters. G, H Higher expression of CST1 was found in GC samples than the matched normal tissues (based on GSE54129 and GSE66229 database). I, J Kaplan–Meier plots of overall survival and progression-free survival for GC samples from the KM Ploter database. K RT-qPCR analysis of CST1 mRNA expression in 100 pairs of GC patient samples. Data are shown as the mean ± SD of triplicate independent sets of experiments; statistical significance was assessed by paired t-test, *<0.05, n = 100. L Western blot analysis was performed using an antibody against CST1 in 5 pairs of GC patients’ samples (upper panel); protein band intensities were measured by ImageJ software and normalized to GAPDH (lower panel). M IHC staining was performed using an antibody against CST1 and representative photographs of CST1 in GC patients. Scale bar: 100 µm. N IHC stain scoring of CST1 in 185 GC tissues and 185 normal tissues, statistical significance was assessed by unpaired t-test, ****<0.0001.

Fig 3: 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).

Fig 4: CST1 mediates GPX4 protein stability to promote migration and invasion in epithelial-mesenchymal transition manner in GC cells.A Western blot analysis of GPX4 in HCG27 and MKN45 stable cell lines transfected with GPX4-siRNA or exogenous overexpressing GPX4. B, C Transwell migration and invasion assays were performed in HGC-27-Vector/HGC-27-CST1 treated with GPX4-siRNA and control as indicated. D, E Transwell migration and invasion assays were performed in MKN45-shNC/MKN45-sh1-CST1/MKN45-sh2-CST1 treated with transient transfection of GPX4 exogenous overexpression or vector control as indicated. Data are presented as the mean ± SD. Statistical significance was assessed by an unpaired t-test. ****p < 0.0001. F Gene set enrichment analysis of CST1 related to invasiveness and degradation of the extracellular matrix. G, H Correlation analysis of CST1 and FN1/Snail in GSE54129 and GSE66229 as illustrated in the dot plot (Person’s correlation test). I Western blot analysis of E-cadherin, Vimetin, FN1 expression in the indicated cells.

Fig 5: The expression of CST1 in NSCLC tumor tissues and adjacent tissues. Immunohistochemical (IHC) staining of CST1 in tumor tissues (A, left: 40 × and right: 200 ×) and adjacent normal tissues (B, left: 40 × and right: 200 ×) from lung adenocarcinoma patients; IHC staining of CST1 in tumor tissues (C, left: 40 × and right: 200 ×) and adjacent tissues (D, left: 40 × and right: 200 ×) from lung squamous cell carcinoma patients; Brown or tan particles are positively stained; E, CST1 protein expression levels were scored in 19 pairs of NSCLC tumor tissues and adjacent tissues; F, Western blotting of CST1 protein expression levels in 4 pairs of NSCLC tumor tissues and adjacent tissues and relative quantification analysis; G, the mRNA relative expression of CST1 in 4 pairs of NSCLC tumor tissues and adjacent tissues. T, the NSCLC tumor tissue; N, the adjacent normal tissue.