Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Expression of Tripartite Motif Containing family across TCGA-ESCA datasets. B Expression profiles of TRIM21 across TCGA datasets. C GO enrichment analysis of TRIM21 correlated genes. D Immunoprecipitation of TRIM21 followed by Coomassie blue staining. E Partial mass spectrometry results following TRIM21 co-immunoprecipitation (co-ip). F Co-immunoprecipitation (co-IP) followed by western blot analysis of TRIM21 and ALKBH5. G GST-Pull down of indicated proteins, demonstrating the molecular binding of TRIM21 and ALKBH5 in vitro. H Plasmids overexpressing ALKBH5-Flag, TRIM21-Myc, and HA-UB were transfected into HEK-293T cells and ubiquitination modification was detected by Western Blot. I Transfection of K63- and K48-linked ubiquitination and indicated plasmids followed by Western Blot analysis. J Transfection of plasmids overexpressing truncated RING domain of TRIM21 and indicated plasmids followed by Western Blot analysis. K Molecular docking and predicted binding sites of TRIM21 and ALKBH5 based on protein sequences obtained from the PDB database. L Mass spectrometry analysis suggests that the ubiquitination site of ALKBH5 may be located at K147. M Transfection of plasmids overexpressing ALKBH5 ubiquitination site mutants and indicated plasmids followed by Western Blot analysis. N Nuclear and cytoplasmic fractionation followed by Western blot analysis showing mutation of ALKBH5's K147 ubiquitination site suppressed ALKBH5 translocation to the nucleus (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Expressing, Immunoprecipitation, Staining, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, In Vitro, Transfection, Ubiquitin Proteomics, Modification, Fractionation, Mutagenesis, Translocation Assay

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Heat map illustrating genes downregulated upon ALKBH5 overexpression detected by RNA sequencing. B KEGG pathway analysis of differentially expressed genes (logFC > 1). C GSEA analysis confirmed significant correlations between RNA degradation pathways and ALKBH5 expression. D RNA Immunoprecipitation (RIP) of ALKBH5 followed by qRT-PCR ( n = 3 independent experiments). E RNA Immunoprecipitation (RIP) of ALKBH5 followed by PCR. F qRT-PCR assays for RNA stability showed that knocking down TRIM21 increased LIAS mRNA stability ( n = 3 independent experiments). G qRT-PCR assays for RNA stability showed that knocking down ALKBH5 increased LIAS mRNA stability ( n = 3 independent experiments). H Measurement of m6A modification levels on LIAS through m6A-IP after knocking down or overexpression of TRIM21 ( n = 3 independent experiments). I Measurement of m6A modification levels on LIAS through m6A-IP after knocking down or overexpression of ALKBH5 ( n = 3 independent experiments). J RIP-qPCR for IGF2BP3 revealed binding of LIAS mRNA to IGF2BP3 ( n = 3 independent experiments). K Knocking down IGF2BP3 notably decreased both the RNA expression levels of LIAS, and this effect was partially rescued by concurrent knockdown of ALKBH5 ( n = 3 independent experiments). L Knocking down IGF2BP3 notably decreased both the protein levels of LIAS, and this effect was partially rescued by concurrent knockdown of ALKBH5 ( n = 3 independent experiments). M qRT-PCR showed LIAS mRNA stability was reduced after IGF2BP3 knockdown ( n = 3 independent experiments). Error bars indicate standard deviations (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Over Expression, RNA Sequencing, Expressing, RNA Immunoprecipitation, Quantitative RT-PCR, Modification, Binding Assay, RNA Expression, Knockdown

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Western blot showing knockdown of TRIM21 significantly increased LIAS expression without affecting ALKBH5 expression. B Nuclear and cytoplasmic fractionation assays followed by Western Blot analysis showed decreased ALKBH5 nuclear localization upon TRIM21 knockdown. C Immunofluorescence staining (IF) demonstrating that knockdown of TRIM21 supresses the nucleocytoplasmic trafficking of ALKBH5. D Apoptosis experiments illustrated that ESCC cells knockdown of TRIM21 or ALKBH5 both increased the sensitivity of cells to cuproptosis, while simultaneous knockout of LIAS attenuated or even counteracted this effect. E CCK8 assays showing that knockdown of TRIM21 inhibited the viability of tumor cells under elesclomol-Cu 2+ stimulation, and this effect could be reversed by LIAS knockdown ( n = 3 independent experiments). F CCK8 assays showing that ESCC cells became more susceptible to elesclomol-Cu 2+ -induced cuproptosis after mutation on ubiquitination site K147 (K147R) compared to Wild Type (WT) ( n = 3 independent experiments). Error bars indicate standard deviations (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Western Blot, Knockdown, Expressing, Fractionation, Immunofluorescence, Staining, Knock-Out, Mutagenesis, Ubiquitin Proteomics

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Subcutaneous xenograft tumor model in nude mice using the esophageal squamous cell carcinoma cell line ECA109, mice were injected with elesclomol in both control (Ctrl) and TRIM21 knockdown (shTRIM21) groups. B Tumor sizes and weights are depicted in the figure ( n = 4 independent experiments). C Proteins from the tumor tissues were extracted for Western Blot analysis, which revealed TRIM21, LIAS protein expression levels. D RNA from the tumor tissues was extracted for qRT-PCR analysis, which revealed TRIM21, LIAS, ALKBH5 mRNA expression levels ( n = 3 independent experiments). E Immunostaining for TRIM21, Ki-67, and TUNEL was performed across the groups. F Quantification of ( E ) ( n = 3 independent experiments). Error bars indicate standard deviations (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Injection, Control, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Immunostaining, TUNEL Assay

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Co-immunoprecipitation assays revealing that OGT and ALKBH5 can interact with each other. B Myc-OGT plasmid, along with HA-UB and Flag-ALKBH5, was transfected into HEK-293T cells, western blot showed overexpression of myc-OGT promoted the ubiquitination of ALKBH5. C Western blot showing co-transfection of TRIM21 with OGT augmented ALKBH5 ubiquitination. D Co-IP followed by western blot showing overexpression of OGT led to an increase in O-glycosylation of ALKBH5. E Co-IP followed by western blot showing knockdown of OGT led to the decrease in the binding of ALKBH5 and TRIM21, also induced upregulation of LIAS. F Co-IP followed by western blot analysis showing reduced ALKBH5 O-glycosylation and interaction with TRIM21 upon introducing the OGT inhibitor OSMI-1 in different concentrations. G A significant negative correlation in protein expression between the co-expression of OGT and TRIM21 with LIAS, Lipoylated-DLAT (Lip-DLAT), and Lipoylated-DLST (Lip-DLST) expression was observed by western blot, upon OSMI-1 treatment, the expressions of Lip-DLAT, Lip-DLST, and LIAS were restored. H Protein expression showing negative correlation between OGT and TRIM21 with LIAS, Lipoylated-DLAT (Lip-DLAT), and Lipoylated-DLST (Lip-DLST) after OGT or TRIM21 knockdown. I Following treatment with a gradient of OSMI-1 concentrations, nuclear-cytoplasmic fractionation experiments showed that OSMI-1 inhibited ALKBH5's accumulation in the nucleus.

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Immunoprecipitation, Plasmid Preparation, Transfection, Western Blot, Over Expression, Ubiquitin Proteomics, Cotransfection, Co-Immunoprecipitation Assay, Glycoproteomics, Knockdown, Binding Assay, Expressing, Fractionation