Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a Pan-cancer analysis of TRIM15 expression. mRNA expression levels (log2(TPM + 1)) of TRIM15 across different tumor types from The Cancer Genome Atlas (TCGA) were shown as box plots, with the midline representing the mean value. b HEK293T cells transfected with the indicated plasmids were subjected to Co-IP. IP samples and whole cell lysates (WCL) were analyzed by Western blot. c Co-IP analysis of the interaction between Flag-TRIM15 and endogenous Axin1 in HCT116 cells. IP samples and WCL were analyzed by Western blot. d Cell lysates derived from HCT116 cells were analyzed by co-IP with control IgG or anti-Axin1 antibody. IP samples and WCL were analyzed by Western blot. e Flag-Axin1, purified from HEK293T cells, was incubated with either immobilized GST or GST-TRIM15, both of which were purified from bacteria. The pulldown samples and input were analyzed by SDS–PAGE followed by Western blot and/or Ponceau S staining. f Immunofluorescence staining of Flag-TRIM15 and GFP-Axin1 in HeLa cells. Nuclei (blue) were counterstained with DAPI. Scale bar represents 20 μm. g Schematic illustration of full-length and truncated Axin1. h Flag-TRIM15, purified from HEK293T cells, was incubated with immobilized GFP-fused Axin1 or its fragments, all of which were also purified from HEK293T cells. The pulldown samples and input were analyzed by SDS–PAGE followed by Western blot. i HEK293T cells transfected with TRIM15-YFP along with either Flag-Axin1 or Flag-Axin1 ΔD plasmids were subjected to Co-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot.

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Control, Purification, Incubation, Bacteria, SDS Page, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a HEK293T cells transfected with the indicated plasmids were subjected to Co-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot. b Flag-Axin1, purified from HEK293T cells, was incubated with either immobilized GST or GST-fused TRIM15 proteins, all of which were purified from bacteria. The pulldown samples and input were analyzed by Western blot and/or Ponceau S staining. c Purified Flag-TRIM15 or Flag-TRIM15 ΔRING proteins were incubated with immobilized GFP-fused Axin1 or E fragment. The pulldown and input samples were analyzed by Western blot. d Immunofluorescence staining of Flag-TRIM15 ΔRING and GFP-Axin1 in HeLa cells. Nuclei (blue) were counterstained with DAPI. Scale bar represents 20 μm. e Purified Flag-TRIM15 or its deletion proteins were incubated with immobilized GFP-Axin1. The pulldown samples and input were analyzed by Western blot. f Purified Flag-Axin1 was incubated with immobilized GST, GST-fused TRIM15, or its deletion proteins. The pulldown and input samples were analyzed by Western blot and/or Ponceau S staining. g Cell lysates from HEK293T cells transfected with the indicated plasmids were boiled in SDS-containing buffer, diluted, and d-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot. h HEK293T cells transfected with Flag-Axin1 with or without GFP-TRIM15. Protein stability was examined by collecting samples at the indicated time after CHX (100 μg/mL) treatment.

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Purification, Incubation, Bacteria, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: SPR (BIAcore) analysis of the dissociation constant ( K d ) for Axin1 ( a ) or TRIM15 ( b ) from immobilized Axin1. Axin1 and Axin1 interaction had a calculated K d of 147 nM, Axin1 and TRIM15 interaction had a calculated K d of 37.8 nM. c HCT116 cells were transfected with control siRNA (siCtrl) or TRIM15 siRNA (siTRIM15), followed by a sucrose density gradient centrifugation experiment to assess the polymerization degree of Axin1 protein. Immunofluorescence staining of Axin1 in DLD-1 cells transfected with siCtrl or TRIM15 siTRIM15 for 48 h. Representative confocal images of endogenous Axin1 ( d ) and the number of Axin1 polymers per cell ( e ). Data are Mean ± SD. Scale bar, 20 μm. f HEK293T cells transfected with the indicated plasmids were subjected to Co-IP. IP and WCL samples were examined by Western blot. HCT116 cells were transfected with siCtrl and siTRIM15 for 72 h, followed by Co-IP using IgG or anti-Axin1 ( g ) or anti-β-TrCP ( h ) antibodies. IP and WCL were analyzed by Western blot. Cells were treated with MG132 (20 μM) for 6 h before lysis. i An in vitro kinase assay was performed by incubation of GST-β-catenin with the indicated proteins purified from HEK293T cells. The samples were analyzed by Western blot. GST-β-catenin was purified from bacteria and shown by Coomassie blue staining.

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Transfection, Control, Gradient Centrifugation, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Lysis, In Vitro, Kinase Assay, Incubation, Purification, Bacteria

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a Immunoblotting of total cell lysates from HCT116 wild type and TRIM15 KO cells treated with DMSO or IWR-1 (50 μM) for 48 h. b AlphaFold prediction of the complex formed by the Axin1 D domain and TRIM15 CC domain. P9715 included an HIV TAT peptide (red) and a peptide derived from the Axin1 D domain. c SPR (BIAcore) analysis of the dissociation constant ( K d ) for P9715 from immobilized TRIM15. The interaction between P9715 and TRIM15 had a calculated K d of 59.2 μM. d HEK293T cells were transfected with the indicated plasmids and then subjected to Co-IP with anti-Flag beads. Cells were treated with P9715 (50 and 100 μM) for 36 h before cell lysis. e HCT116 cells were treated with P9715 (100 μM) for 48 h, followed by the sucrose density gradient centrifugation experiment to assess the polymerization degree of Axin1 protein. f Immunoblotting of total cell lysates derived from HCT116 cells treated with IWR-1 (50 μM), P9715 (100 μM), or their combination for 72 h. Xenograft tumor formation by HCT116 cells in nude mice that were treated with IWR-1, P9715, or their combination. The tumor volume over time ( g ) tumor images ( h ) and tumor weight ( n = 5 animals) ( i ) were shown. j The proposed model. Data are Mean ± SD. One-way ANOVA for ( g , i ).

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Western Blot, Derivative Assay, Transfection, Co-Immunoprecipitation Assay, Lysis, Gradient Centrifugation