Journal: The Journal of Biological Chemistry

Article Title: CAPN15 is a non-proteasomal, ubiquitin-directed calpain protease that regulates cell adhesion by cleaving E-cadherin

doi: 10.1016/j.jbc.2025.111034

Figure Lengend Snippet: CAPN15 recognizes the ubiquitinated cadherin-catenin complex via the NZF domains. A , differential interference contrast images of HCT116, KO, WTtg, CStg, and NEtg cells. Scale bar, 20 μm. B , the mean log 2 fold change of the protein abundance (CStg versus NEtg immunoprecipitates) with -log 10 p value of the interactome was shown on the x- and y-axes, respectively. Significantly increased proteins were defined as those with the value of the log 2 fold change >1 with p value < 0.05 (plots in a colored area). It should be noted that there was no change in abundance of CAPN15 in the CStg and NEtg immunoprecipitates. C , the heatmap shows the relative abundance of cadherins and catenins in the KO, WTtg, CStg, and NEtg immunoprecipitates. D , KO, WTtg, CStg, and NEtg cells were treated with dimethyl sulfoxide (DMSO; vehicle) or 1 μM MLN7243. Cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody, followed by western blotting. Asterisks indicate nonspecific bands. Vertical lines indicate the ubiquitinated forms. E , immunoprecipitated fraction from DMSO (vehicle)-treated CStg cells was incubated with or without ubiquitin-specific protease 2 (USP2cc), followed by western blotting. The asterisk indicates the non-specific band. Vertical lines indicate the ubiquitinated forms.

Article Snippet: Anti-FLAG immunoprecipitate prepared from CStg cells was incubated with 1 μM USP2 catalytic domain (USP2cc; R&D Systems) for 1 h at 37 °C.

Techniques: Quantitative Proteomics, Immunoprecipitation, Western Blot, Incubation, Ubiquitin Proteomics

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A The ingenuity pathway analysis (IPA). Analysis of proteomics in the kidney of WT and U2KO mice. B Volcano plot of differentially expressed genes in the kidney from the kidneys of U2KO and WT mice ( P < 0.05). Genes related to the TGFβ signaling pathways are labeled. C , D WT or U2KO TECs incubated with CHX (C ) or MG132 ( D ) as indicated. The total cell lysates were prepared and western blotting was performed using the indicated antibodies. E Co-IP assays were performed using lysates from WT TECs stimulated with TGF-β1, and western blotting using the indicated antibodies. F WT TECs transfected with Ad HA- Txndc5 and/or Ad-Flag Usp2 as indicated, and then subjected to TGF-β1 stimulation. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. G HK2 cells transfected with Ad-Flag USP2 WT (Flag-WT) or Ad-Flag USP2 C276A. Total cell lysates were subjected to Co-IP with anti-TXNDC5 antibody, and western blotting using the indicated antibodies. H , I The database provides TXNDC5 posttranslational modification of individual sites ( H ). HK2 or shUSP2 cells transfected with Ad-Flag TXNDC5 WT, K118R, K143R, K150R, and K244R as indicated, and the total cell lysates were prepared and western blotting using the indicated antibodies ( I ). J HK2 transfected with siRNA USP2 (si USP2 ), Ad-Flag TXNDC5 WT, or Ad-Flag TXNDC5 K150R as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies. K USP2-TXNDC5 docking with the HDOCK server. High magnification of the boxed areas is presented on the left. The arrow indicates K150 of the TXNDC5 protein. L WT TECs were transfected with Ad- Usp2 and/or si Txndc5 as indicated, and the total cell lysates were prepared and western blotting using the indicated antibodies. M HK2 transfected with Ad- Tnxdc5 and/or si Usp2 as indicated, and the total cell lysates were prepared and western blotting using the indicated antibodies (left). WT or S2KO mice were transfected with AAV-ctrl or AAV-sh Txndc5 , and then subjected to UUO surgery. Total cell lysates from the kidney of mice were prepared, and western blotting was performed using the indicated antibodies (right). N Schematic illustration of TXNDC5 deletion mutants (left). The numbers indicate the amino acid positions. HA- TXNDC5 deletion mutants were coexpressed with Flag- USP2 in HEK293T cells. O – S HK2 cells were transfected with Ad-Null or Ad- USP2 for 24 h, and treated with or without 2 ng/ml TGFβ1 for the indicated times. Representative images of SMAD3 immunofluorescence staining are shown in ( O ; Scale bar = 25 μm), and densitometry quantification of nuclear levels of SMAD3 are shown in ( P ; elements in this figure were created with BioRender.com). Q Western blot analyses of SMAD3 in the fractions extracted from TECs. R 3TP-Lux luciferase activity assay in HEK293T cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and Ad-null or Ad- USP2 for 24 h, followed by the treatment with or without 2 ng/ml TGFβ1 for 16 h. Relative luciferase activity is presented as folds of that in the cells with transfection of Ad-null ( n = 5). S HK2 were transfected with Ad-null or Ad USP2 for 24 h, and treated with or without 2 ng/ml TGFβ1 for 24 h. Western blot analysis of TGFBR1, USP2, SMAD3, pSMAD3, and Tubulin in HK2 cells (left). For all panels, data were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Protein-Protein interactions, Labeling, Incubation, Western Blot, Co-Immunoprecipitation Assay, Transfection, Modification, Immunofluorescence, Staining, Luciferase, Activity Assay, Plasmid Preparation

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A The IPA Analysis of proteomics in the kidney of WT and U2KO mice. B , C WT TECs transfected with siCtrl or si Usp2 as indicated in the present with or without TGFβ1. B The total cell lysates were prepared and western blotting was performed using the indicated antibodies. C MDA, SOD, GSH, and Fe 2+ levels in cell homogenates were detected by a commercial kit ( n = 5). D , E WT TECs transfected with Ad-null or Ad Usp2 as indicated in the presence or absence of TGFβ1. D The total cell lysates were prepared and western blotting using the indicated antibodies. E MDA, SOD, GSH, and Fe 2+ levels in cell homogenates were detected by commercial kit ( n = 5). F Representative images of BODIPY 581/591 C11 staining. Scale bar = 50 μm. G WT TECs transfected with Ad Usp2 and si Tfrc as indicated upon TGFβ1 stimulation. (Left) The total cell lysates were prepared and western blotting using the indicated antibodies. (Right) MDA, SOD, GSH, and Fe 2+ levels in cell homogenates were detected by a commercial kit ( n = 5). H WT TECs transfected with si Usp2 and Ad Tfrc as indicated upon TGFβ1 stimulation. (Left) The total cell lysates were prepared and western blotting using the indicated antibodies. (Right) MDA, SOD, GSH, and Fe 2+ levels in cell homogenates were detected by a commercial kit ( n = 5). I , J WT or U2KO TECs incubated with CHX ( I ) or MG132 ( J ) as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies. K Co-IP assays were performed using lysates from WT TECs stimulated with TGF-β1, and western blotting using the indicated antibodies. L WT TECs transfected with Ad HA- Tfrc and/or Ad-Flag Usp2 as indicated, and then subjected to TGF-β1 stimulation. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. M HK2 cells transfected with Ad-Flag USP2 WT (Flag-WT) or Ad-Flag USP2 C276A. Total cell lysates were subjected to Co-IP with anti-TFRC antibody, and western blotting using the indicated antibodies. N The database provides TXNDC5 posttranslational modification of individual sites (left). HK2 cells transfected with sh USP2 , Ad-Flag TFRC WT, K39R and K160R as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies (right). O HK2 cells transfected with siRNA USP2 (si USP2 ), Ad-Flag TFRC WT, or Ad-Flag TFRC K39R as indicated. The total cell lysates were prepared and western blotting using the indicated antibodies. P Schematic illustration of TFRC deletion mutants (left). The numbers indicate the amino acid positions. HA- TFRC deletion mutants were coexpressed with Flag- USP2 in HEK293T cells (right). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Transfection, Western Blot, Staining, Incubation, Co-Immunoprecipitation Assay, Modification

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A RT-PCR ( n = 5) and western blot analysis ( n = 3) of the expression of USP2 in selected mouse renal cells, including mouse podocytes (MPC), rat glomerular endothelial cells (GEC), mouse TECs, and mouse fibroblast (MF). B Single-cell sequencing results show that USP2 is predominantly expressed in renal tubular cells. C – I Control (AAV-Ctrl) or Usp2 OE (AAV- Ksp - Usp2 ) mice were randomly assigned to sham or UUO surgery according to an established protocol ( n = 6). C Total cell lysates from the kidney of mice subjected to Co-IP with anti-TFRC or anti-TXNDC5 antibody, and western blotting using the indicated antibodies. D Representative images of Masson’s trichrome staining and Sirius red in kidneys from mice (scale bar, 100 μm). E Western blot analysis of the expression of CCN2, FN1, COL1A1, COL3A1, GPX4, HMOX1, and Tubulin. F The mRNA level of Col1a1, Fn1, Eln, Ccn2, Acta2 , Col3a1, Ngal , and Kim-1 in the kidney of mice. G , H The level of Iron, MDA, SOD, GSH, and the ratio of GSSG in renal homogenate. I Representative TUNEL staining and quantification (three field per mice; n = 6) of apoptotic cells in kidney sections from mice (Scale bar = 20 μm). For all panels, data were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Sequencing, Control, Co-Immunoprecipitation Assay, Staining, TUNEL Assay

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A – G WT or U2TKO mice were randomly assigned to sham or UUO surgery according to an established protocol. Kidney samples were obtained from mice on day 10 post-UUO or sham surgery ( n = 6 per group). A Representative images of Masson’s trichrome staining and Sirius red in kidneys from mice (scale bar = 100 μm). B Western blot analysis of the expression of USP2, CCN2, FN1, COL1A1, COL3A1, GPX4, HMOX1, and Tubulin. C , D The mRNA level of Col1a1, Fn1, Eln, Ccn2, Acta2 , Col3a1, Ngal , and Kim-1 . E The level of Iron, MDA, SOD, GSH, and the ratio of GSSG in renal homogenate ( n = 6). F Representative TUNEL staining and quantification (3 field per mice; n = 6) of apoptotic cells in kidney sections from mice (Scale bar = 20 μm). G , H Total cells lysates from kidney of mice subjected to Co-IP with anti-USP2, anti-TFRC, or anti-TXNDC5 antibody, and western blotting using indicated antibodies. For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Staining, Western Blot, Expressing, TUNEL Assay, Co-Immunoprecipitation Assay

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A Total cell lysates from WT TECs subjected to Co-IP with anti-SIRT2 or anti-USP2 antibody, and western blotting using indicated antibodies (left). HK2 cells were transfected with Ad HA- SIRT2 and/or Ad-Flag USP2 as indicated. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies (right). B WT TECs transfected with Ad-null or Ad- Sirt2 as indicated, followed by TGF-β1 stimulation. Total cell lysates were subjected to Co-IP with anti-USP2 antibody, and western blotting using the indicated antibodies. C HK2 cells transfected with Ad-Flag Usp2 WT, K139R, K205R, K241R, K340R, K420R, K447R, K464R, or K473R as indicated. The total cell lysates were prepared and western blotting using indicated antibodies. D The conservation of USP2 K447 in different species. E WT TECs transfected with Ad-Flag Usp2 WT and then incubated with TGFβ1 for the indicated time. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. F Total cells lysates from the kidney of UUO or FA-treated mice subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. G WT TECs transfected with Ad-Flag Usp2 WT or Ad-Flag Usp2 K447R in the presence or absence of TGFβ1. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. H – L WT TECs transfected with Ad-null, Ad- Usp2 WT, and Ad- Usp2 K447R under TGFβ1 stimulation was indicated. Representative images of SMAD3 immunofluorescence staining are shown in ( H ; (Scale bar = 25 μm)), and densitometry quantification of nuclear levels of SMAD2 are shown in ( I ). J Western blot analyses of SMAD3 in the fractions extracted from TECs. K 3TP-Lux luciferase activity assay in HEK293T cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and Ad-null, Ad- USP2, or Ad- USP2 K447R for 24 h, followed by the treatment with or without 2 ng/ml TGFβ1 for 16 h. Relative luciferase activity is presented as folds of that in the cells with transfection of Ad-null ( n = 5). L Western blot analysis of TGFBR1, USP2, SMAD3, pSMAD3, and Tubulin in TECs (left). The mRNA level of Col1a1 , Col1a2 , Serpine1 , and Smad7 as determined by qPCR (right; n = 5). M WT TECs transfected with Ad-Flag- Usp2 and Ad- Sirt2 or si Sirt2 in the presence or absence of TGFβ1 as indicated. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. N WT TECs transfected with Ad HA- Sirt2 or Ad-Flag- Usp2 under TGFβ1 stimulation as indicated. Total cell lysates were subjected to Co-IP with anti-HA antibody, and western blotting using the indicated antibodies. O WT TECs transfected with Ad-Flag- Usp2 WT, Ad-Flag- Usp2 K447R, or Ad- Sirt2 under TGFβ1 stimulation as indicated. Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. P HK2 cells transfected with Ad-Flag- USP2 WT, Ad-Flag- SIRT2 H187Y, or Ad HA- SIRT2 under TGFβ1 stimulation as indicated (Elements in this figure were created with BioRender.com). Total cell lysates were subjected to Co-IP with anti-Flag antibody, and western blotting using the indicated antibodies. Q USP2-SIRT2 docking with the HDOCK server. High magnification of the boxed areas is presented on the left. The arrow indicates K447 of the USP2 protein. R Total cells lysates from the kidney of UUO and FA-treated WT or S2KO mice subjected to Co-IP with anti-USP2 antibody, and western blotting using the indicated antibodies. For all panels, data were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Incubation, Immunofluorescence, Staining, Luciferase, Activity Assay, Plasmid Preparation

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A – H WT and Usp2 KO (U2KO) mice were transfected with AAV-ctrl, AAV-Ksp-WT Usp2 , and AAV-Ksp- Usp2 K447R, and after 2 weeks of transfection, mice were randomly assigned to UUO surgery according to an established protocol ( n = 6). A Representative images of Masson’s trichrome staining and Sirius red in kidneys from mice (scale bar, 100 μm). B Western blot analysis of the expression of CCN2, FN1, COL1A1, COL3A1, GPX4, HMOX1, and Tubulin. C , D The mRNA level of Col1a1, Fn1, Eln, Ccn2, Acta2 , Col3a1, Ngal , and Kim-1 in the kidney of mice. E The level of Iron, MDA, SOD, GSH and the ratio of GSSG in renal homogenate. F Representative TUNEL staining and quantification (3 field per mice; n = 6) of apoptotic cells in kidney sections from mice (Scale bar = 20 μm). G , H Total cells lysates from kidney of mice subjected to Co-IP with anti-TFRC or anti-TXNDC5 antibody, and western blotting using indicated antibodies. For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with Bonferroni correction test. For all panels, data were presented as mean ± SD. ** P < 0.01, *** P < 0.001 by one-way ANOVA with Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Transfection, Staining, Western Blot, Expressing, TUNEL Assay, Co-Immunoprecipitation Assay

Journal: Communications Biology

Article Title: Lactate orchestrates the TGFβ pathway and ferroptosis nexus in organ fibrosis via USP2 lactylation

doi: 10.1038/s42003-025-09286-z

Figure Lengend Snippet: A – G WT mice were randomly assigned to sham or UUO surgery according to an established protocol, and after 3 days, mice were treated with ML364 (10 mg/kg body weight, i.v. daily) or vehicle. Kidney samples were obtained from mice on day 10 post-UUO or sham surgery ( n = 6 per group). A Representative images of Masson’s trichrome staining and Sirius red in kidneys from mice (scale bar = 100 μm). B Western blot analysis of the expression of USP2, CCN2, FN1, COL1A1, COL3A1, GPX4, HMOX1, and Tubulin. C , D The mRNA level of Col1a1, Fn1, Eln, Ccn2, Acta2 , Col3a1, Ngal , and Kim-1 . E The level of Iron, MDA, SOD, GSH and the ratio of GSSG in renal homogenate. F Representative TUNEL staining and quantification (3 field per mice; n = 6) of apoptotic cells in kidney sections from mice (Scale bar = 20 μm). G , H Total cell lysates from the kidney or liver of mice subjected to Co-IP with anti-TFRC or anti-TXNDC5 antibody, and western blotting using the indicated antibodies. For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with Bonferroni correction test.

Article Snippet: Mice were divided into groups ( n = 6 per group) receiving AAV9-empty vector (AAV9-Ctrl), AAV9- Ksp - Usp2 ( Usp2 OE group), AAV9- Ksp - Usp2 K447R (mutant group), and AAV9- Ksp -short hairpin RNA targeting Usp2 (AAV-shUsp2); all AAV constructs were provided by GeneChem (Shanghai, China).

Techniques: Staining, Western Blot, Expressing, TUNEL Assay, Co-Immunoprecipitation Assay