Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 Nov 9;43(1):123–149. doi: 10.1002/cac2.12386

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2022 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat‐sen University Cancer Center.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

PMC Copyright notice

graphic file with name CAC2-43-123-g002.jpg — TRIM21 was the E3 ubiquitin ligase of STAT1 and TGM2 promoted the dissociation of TRIM21 and STAT1 (A) TRIM21 was knocked down in GC cells by transfecting siRNAs. And western blotting confirmed that TRIM21 si‐1 showed the highest inhibitory efficacy on TRIM21 protein. What's more, TRIM21 si‐1 increased STAT1 protein levels in GC cells. The bar chart showed the quantitative analysis of TRIM21 and STAT1 protein levels. (B) TRIM21 was knocked down in GC cells by transfecting siRNAs. The knockdown efficacy was evaluated by qPCR, and the results showed that TRIM21 si‐1 showed the highest inhibitory efficacy on TRIM21 mRNA. (C) The effect of TRIM21 knockdown on STAT1 mRNA levels was examined by qPCR, and the results showed that TRIM21 knockdown had no effect on STAT1 mRNA levels. (D) GC cells were transiently transfected with Ub‐HA plasmids to examine the effect of TRIM21 knockdown on the ubiquitin level of STAT1 by Co‐IP and western blotting. The results showed that TRIM21 knockdown reduced the ubiquitin level of the exogenous and endogenous STAT1 (E) TRIM21 expression in TGM2‐overexpressing/TGM2‐knocked down GC cells and NC cells were evaluated by western blotting and qPCR. The results showed that TGM2 had no effect on TRIM21 expression at both the mRNA and protein levels. (F) The colocalization of TRIM21 and STAT1 was examined using immunofluorescence. The results showed that TGM2 overexpression/knockdown upregulated/downregulated STAT1 levels, while had no effect on TRIM21 level in GC cells. TGM2 expression didn't affect the colocalization capacity of TRIM21 and STAT1. The representative images of immunofluorescence were displayed. The upper bar chart showed the quantitative analysis of STAT1 and TRIM21 protein levels, *: P<0.05. The lower bar chart showed the quantitative analysis of colocalization ratio of TRIM21 and STAT1. (G) Co‐IP and western blotting showed that TGM2 facilitated the dissociation of TRIM21 and STAT1. And TGM2 was not a component in the Co‐IP mixture. Bar chart showed the quantitative analysis of TRIM21 and STAT1 protein levels. (H) The role of TRIM21 in the prognosis of patients with GC was analyzed with an online bioinformatics tool (The Kaplan‐Meier plotter), and the result indicated that TRIM21 showed anti‐tumor properties, which prolonged the postoperative OS (P = 0.0056) and first progression (P = 0.0025) in patients with GC. Abbreviations: GC, gastric cancer; TGM2, transglutaminase 2; v, vector; NC, negative control; OS, overall survival; FP, first progression; Co‐IP, co‐immunoprecipitation; IP, immunoprecipitation.

graphic file with name CAC2-43-123-g006.jpg — TRIM21 was the E3 ubiquitin ligase of STAT1 and TGM2 promoted the dissociation of TRIM21 and STAT1 (A) TRIM21 was knocked down in GC cells by transfecting siRNAs. And western blotting confirmed that TRIM21 si‐1 showed the highest inhibitory efficacy on TRIM21 protein. What's more, TRIM21 si‐1 increased STAT1 protein levels in GC cells. The bar chart showed the quantitative analysis of TRIM21 and STAT1 protein levels. (B) TRIM21 was knocked down in GC cells by transfecting siRNAs. The knockdown efficacy was evaluated by qPCR, and the results showed that TRIM21 si‐1 showed the highest inhibitory efficacy on TRIM21 mRNA. (C) The effect of TRIM21 knockdown on STAT1 mRNA levels was examined by qPCR, and the results showed that TRIM21 knockdown had no effect on STAT1 mRNA levels. (D) GC cells were transiently transfected with Ub‐HA plasmids to examine the effect of TRIM21 knockdown on the ubiquitin level of STAT1 by Co‐IP and western blotting. The results showed that TRIM21 knockdown reduced the ubiquitin level of the exogenous and endogenous STAT1 (E) TRIM21 expression in TGM2‐overexpressing/TGM2‐knocked down GC cells and NC cells were evaluated by western blotting and qPCR. The results showed that TGM2 had no effect on TRIM21 expression at both the mRNA and protein levels. (F) The colocalization of TRIM21 and STAT1 was examined using immunofluorescence. The results showed that TGM2 overexpression/knockdown upregulated/downregulated STAT1 levels, while had no effect on TRIM21 level in GC cells. TGM2 expression didn't affect the colocalization capacity of TRIM21 and STAT1. The representative images of immunofluorescence were displayed. The upper bar chart showed the quantitative analysis of STAT1 and TRIM21 protein levels, *: P<0.05. The lower bar chart showed the quantitative analysis of colocalization ratio of TRIM21 and STAT1. (G) Co‐IP and western blotting showed that TGM2 facilitated the dissociation of TRIM21 and STAT1. And TGM2 was not a component in the Co‐IP mixture. Bar chart showed the quantitative analysis of TRIM21 and STAT1 protein levels. (H) The role of TRIM21 in the prognosis of patients with GC was analyzed with an online bioinformatics tool (The Kaplan‐Meier plotter), and the result indicated that TRIM21 showed anti‐tumor properties, which prolonged the postoperative OS (P = 0.0056) and first progression (P = 0.0025) in patients with GC. Abbreviations: GC, gastric cancer; TGM2, transglutaminase 2; v, vector; NC, negative control; OS, overall survival; FP, first progression; Co‐IP, co‐immunoprecipitation; IP, immunoprecipitation.