Journal: Cell Death & Disease

Article Title: DJ-1 counteracts Caveolin-1-mediated necroptosis to inhibit epithelial barrier dysfunction in colitis

doi: 10.1038/s41419-025-07989-z

Figure Lengend Snippet: A Representative IHC analysis of DJ-1 expression in colonic tissue samples from healthy controls and patients with UC or CD (magnification ×200; scale bar = 50 µm). B Statistical analysis of DJ-1 expression in the healthy controls ( n = 13), patients with UC ( n = 10) or CD ( n = 11). C Correlation analysis of the CAV1 and DJ-1 IHC staining IOD/area score is shown ( n = 36). D HCT116 cells were infected with a FLAG-tagged CAV1 overexpression plasmid or the empty vector (vehicle), and total FLAG-tagged CAV1 was immunoprecipitated. E Endogenous co-IP: HEK-293 cell lysates were immunoprecipitated with anti–DJ-1 or control IgG antibodies. F Endogenous co-IP: HT29 cell lysates were immunoprecipitated with anti–DJ-1 or control IgG antibodies. G Representative images of immunofluorescence staining of IBD human colon sections (DJ-1: green, CAV1: red, DAPI nuclear: blue, magnification ×600). H Western blot analysis of colonic CAV1 protein levels in the WT and DJ-1 KO mice with DSS-induced colitis. I Quantitative analysis of the above CAV1 protein levels ( n = 4). J HEK293 cells infected with FLAG-tagged CAV1, HA-tagged DJ-1 plasmid and empty vector were stimulated with 100 ng/ml TNF-α and 25 µM zVAD-fmk for 24 h and then treated with MG132 (25 µM, I) for another 6 h. All data are the means ± SD. * p < 0.05, *** p < 0.001, **** p < 0.0001, two-tailed.

Article Snippet: For the necroptosis inhibition experiments, WT and DJ-1 KO mice were treated with 2 mg/mL GSK’872 (MCE, USA) every two days or 1 mg/mL GW806742X (MCE, USA) each day by intraperitoneal injection under a 7-day DSS administration.

Techniques: Expressing, Immunohistochemistry, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Immunofluorescence, Staining, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DJ-1 counteracts Caveolin-1-mediated necroptosis to inhibit epithelial barrier dysfunction in colitis

doi: 10.1038/s41419-025-07989-z

Figure Lengend Snippet: WT, DJ-1 KO, CAV1 KO and DKO mice were treated with DSS for 7 days. WT NC = 5, WT DSS = 12, DJ-1 KO DSS = 12, CAV1 KO DSS = 6, DKO DSS = 5. Body weight change ( A ), survival rates ( B ) and the disease activity index ( C ) were monitored daily. D Mouse colon lengths were measured after sacrifice. E Intestinal permeability was evaluated by measuring the concentration of FITC-dextran in the blood serum. Histological scores ( F ) were determined in a double-blinded manner, and the histological analysis of colon tissue samples is shown ( G ) (upper: magnification ×100; scale bar = 100 µm, lower: magnification ×400; scale bar = 20 µm). H Quantitative PCR analysis was used to assess cytokine and chemokine production in whole-colon homogenates. DAI scores and inflammation scores are expressed as median and IQR. Other All data are the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns no significant, two-tailed.

Article Snippet: For the necroptosis inhibition experiments, WT and DJ-1 KO mice were treated with 2 mg/mL GSK’872 (MCE, USA) every two days or 1 mg/mL GW806742X (MCE, USA) each day by intraperitoneal injection under a 7-day DSS administration.

Techniques: Activity Assay, Permeability, Concentration Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DJ-1 counteracts Caveolin-1-mediated necroptosis to inhibit epithelial barrier dysfunction in colitis

doi: 10.1038/s41419-025-07989-z

Figure Lengend Snippet: A Representative IHC analysis of p-RIPK1 expression in colonic tissue samples (IHC upper: magnification ×100；scale bar = 100 µm, lower: magnification ×400；scale bar = 20 µm). B Quantification of IHC analysis of p-RIPK1 (WT NC = 5, WT DSS = 6, DJ-1 KO DSS = 7, CAV1 KO DSS = 4, DKO DSS = 5) expression. C Western blotting was used to analyze CAV1, DJ-1 and necroptosis signaling pathway molecule protein levels in colon tissue samples from the mice treated as described above. D Intestinal organoids from the WT, DJ-1 KO, CAV1 KO and DKO mice treated as indicated with the combination of TNF-α (100 ng/ml) and zVAD-fmk (25 µM) (TZ), or TZ+ necrostatin-1 (Nec-1, 30 µM) for 12 h and stained with PI (red) for 24 h (magnification ×200；scale bar = 50 µm). E Quantification of PI intensities. n = 12 organoids from 3 mice per group. F DJ-1 and CAV1 expression were knocked down in HCT116 cells, and the cells were stimulated with TZ for 24 h. G Western blot analysis of DJ-1 and CAV1 overexpression in the TZ-stimulated HCT116 cells. All data are the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-tailed.

Article Snippet: For the necroptosis inhibition experiments, WT and DJ-1 KO mice were treated with 2 mg/mL GSK’872 (MCE, USA) every two days or 1 mg/mL GW806742X (MCE, USA) each day by intraperitoneal injection under a 7-day DSS administration.

Techniques: Paraffin-embedded Immunohistochemistry, Expressing, Western Blot, Staining, Over Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DJ-1 counteracts Caveolin-1-mediated necroptosis to inhibit epithelial barrier dysfunction in colitis

doi: 10.1038/s41419-025-07989-z

Figure Lengend Snippet: WT and DJ-1 KO mice were treated with 2 mg/ml GSK’872 every two days by intraperitoneal injection after 7 days of DSS administration. WT DSS = 7, WT DSS + GSK’872 = 9, DJ-1 KO DSS = 7, DJ-1 KO DSS + GSK’872 = 6. Body weight change ( A ), survival rates ( B ) and the DAI scores ( C ) were scored daily. Mice were sacrificed on Day 7, and colon lengths ( D ) were measured. E HE staining in colon tissue samples from the WT and DJ-1 KO mice is shown (magnification ×100; scale bar=100 µm). F Semiquantitative histopathological scoring was performed. G Quantitative PCR analysis of cytokines and chemokines in colons from the WT and DJ-1 KO DSS-treated mice. The WT and DJ-1 KO mice were treated with 1 mg/ml GW806742X (GW) for each day by intraperitoneal injection under a 7-day DSS administration. WT DSS = 5; WT DSS + GW = 7; DJ-1 KO + GW = 7. Body weight change ( H ), survival rates ( I ) and DAI scores ( J ) were scored daily. Colon lengths ( K ) were measured. H&E staining ( L ) in colon tissue samples is shown, and semiquantitative scoring of histopathology ( M ) was performed (upper: magnification ×40; scale bar = 500 µm, lower: magnification ×100; scale bar=200 µm). N Quantitative PCR analysis of cytokines and chemokines in colons. DAI scores and inflammation scores are expressed as median and IQR. Other All data are the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-tailed.

Article Snippet: For the necroptosis inhibition experiments, WT and DJ-1 KO mice were treated with 2 mg/mL GSK’872 (MCE, USA) every two days or 1 mg/mL GW806742X (MCE, USA) each day by intraperitoneal injection under a 7-day DSS administration.

Techniques: Injection, Staining, Real-time Polymerase Chain Reaction, Histopathology, Two Tailed Test

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: The 22 DEPRGs in DN samples compared to normal samples.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques:

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: Validation of Hub DEPRGs in DN. ( A ) Expression box plot of the 5 Hub DEPRGs in the validation dataset GSE142025. ( B ) Expression box plot of the 5 Hub DEPRGs in the validation dataset GSE96804. The blue bars represent control specimens from normal individuals, and the red bars represent specimens from DN patients. ( C–F ) ROC curves of CASP1 ( C ), TXNIP ( D ), IRF9 ( E ) and TRAF3 ( F ) in the training dataset GSE30529. ROC curves the represents receiver operator characteristic curves; AUC represents area under the curve; CI represents credibility interval.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques: Biomarker Discovery, Expressing, Control

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: Correlation between immune cells and Hub DEPRGs. Correlation between CASP1 ( A ), TXNIP ( B ), IRF9 ( C ), TRAF3 ( D ) and infiltrating immune cells in DN and normal sample groups.The size of the dots from small to big represents the degree of correlation coefficient from low to high and the colour of the dots from yellow to green represents the p -value from high to low. The right side of the numbers depict p -value, with red color indicating a p -value less than 0.05.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques:

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: Establishment of DN model rats and validation of the ceRNA networks. ( A ) UTP, SCr and BUN levels in rats. ( B ) Pathologic morphology of rat kidney tissues observed by HE, PAS and TUNEL staining. ( C ) Protein levels of NLRP3, pro-casp1, cleaved-casp1, GSDMD, GSDMD-N, IRF9, TXNIP and TRAF3 by Western blotting, and quantitative analysis in rat kidney tissues (The eight target gene bands originated from different gels, and the gels/blots were cropped. Samples derive from the same experiment and that gels/blots were processed in parallel. Original blots/gels are presented in Supplementary Fig. S2). ( D ) The relative expression of mRNAs and miRNAs in rat kidney tissues was detected by qRT-PCR. P value was shown as: * p < 0.05; ** p < 0.01.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques: Biomarker Discovery, TUNEL Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Bioinformatics analysis and validation of novel biomarkers and competitive endogenous RNA networks involved in pyroptosis in diabetic nephropathy

doi: 10.1038/s41598-025-87854-3

Figure Lengend Snippet: qRT-PCR primers information.

Article Snippet: The blots were probed with primary antibodies for GAPDH (1:5000, Proteintech, 10494-1-AP), NLRP3 (1:1500, Proteintech, 27458-1-AP), pro-casp1 (1:1500, Affinity, AF5418), cleaved-casp1 (1:1000, Affinity, AF4005), GSDMD (1:1500, Affinity, AF4012), and GSDMD-N (1:1500, Affinity, AF4012), IRF9(1:1000, Proteintech, 14167-1-AP) , TXNIP (1:1000, Proteintech, 18243-1-AP), TRAF3(1:3000, Proteintech, 18099-1-AP).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Identification of a Novel Serum Biomarker for Tuberculosis Infection in Chinese HIV Patients by iTRAQ-Based Quantitative Proteomics

doi: 10.3389/fmicb.2018.00330

Figure Lengend Snippet: Differentially expressed proteins in the sera of HIV positive patients with active tuberculosis (TB) relative to controls (only HIV positive patients).

Article Snippet: The concentrations of ENG (CUSABIO, CSB-E10030h, China), PSMB2 (CUSABIO, CSB-E17836h, China), HSP90AA1 (CUSABIO, CSB-E13462h, China), HSPA8 (CUSABIO, CSB-EL010829HU, China), CHI3L1 (CUSABIO, CSB-E13608h, China), CAP1 (CUSABIO, CSB-EL004486HU, China), ORM1 (CUSABIO, CSB-EL017237HU, China), RETN (CUSABIO, CSB-E06884h, China), LPA (CUSABIO, CSB-EQ028005HU, China) and ZYX (CUSABIO, CSB-EL027165HU, China) in the serum samples were quantified using ELISA according to the manufacturer’s instructions.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 is positively correlated with favorable prognosis in breast cancer. ( a ) High TRAF3 mRNA expression levels are associated with better OS (Living vs. Diseased, Mann–Whitney U test), lower disease stage (Bonferroni correction), lower lymph node stage (N) (N0 vs. N1: p = 0.004, Bonferroni correction) and lower tumor stage (T) (T1 vs. T3: p = 0.019, Bonferroni correction) in the TCGA-BRCA cohort. ( b ) High TRAF3 mRNA expression presents with a statistically significant better OS ( p = 0.00405) and DMFS ( p = 0.00729) in the ER-negative breast cancer cohort employed by GOBO, with ER-positive disease presenting a similar association despite not reaching statistical significance.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Forced TRAF3 expression in breast cancer cell lines induces partial EMT and affects cell proliferation. ( a ) Invasion, migration and colony formation assays depicting an opposing phenotype between migratory and proliferative states of MCF7-TRAF3 cells. ( b ) Western blot analyses for the indicated proteins in MDA-MB-231 and MCF-7 cells (control and TRAF3 expressing). ( c ) ICC for the indicated proteins in MCF-7 cells, indicating significant downregulation of key molecules upon TRAF3 expression (ns: no significance; *** p < 0.001 Student’s t -test).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Migration, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Identification of interactors, pathways and processes of TRAF3 in breast cancer. ( a ) Volcano plot of significant TRAF3 interactions in MCF-7 cells (FDR < 0.05). ( b ) Top 20 enriched pathways (Metascape) among proteins that interact with TRAF3 in MCF-7 cells with −log10(Padj) > 10 −20 . ( c ) Significantly enriched pathways among genes co-expressed with TRAF3 in the TCGA BRCA cohort. ( d ) Representative BRCA cases from the TCGA cohort presenting with High and Low TILs (upper panel). High TRAF3 mRNA expression is correlated ( p = 0.02, Mann–Whitney U Test) with High stromal TILs in the TCGA BRCA cohort ( n = 200).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression across cell populations in the scRNA human breast cancer dataset. ( a ) UMAP visualization of 81,389 quality-filtered single cells derived from the Breast Cancer Atlas, colored by cell type annotation. ( b ) Feature plot showing log-normalized TRAF3 expression projected onto the UMAP embedding. ( c ) Violin plots depicting log-normalized TRAF3 expression across each of the cell types. Statistical comparisons were performed using Wilcoxon rank-sum tests, comparing each cell type against all remaining cells, followed by Benjamini–Hochberg correction for multiple testing. Asterisks (*) indicate adj p -values < 0.05. ( d ) Volcano Plot of Differential expression of TRAF3 -positive ( TRAF3 +) vs. negative ( TRAF3 -) Cancer Epithelial (CE) cells. The x-axis represents the log 2 fold change of expression in TRAF3 -positive versus TRAF3 -negative cells, and the y-axis shows the −log 10 adjusted p -value (FDR). Points are colored according to FDR significance, while labels highlight specific immunologically relevant genes, colored according to the following categories: (i) Immunogenicity—Immunogenicity/Antigen Presentation; (ii) MHC-I—MHC class I pathway (CD8 + T-cell recognition); (iii) MHC-II—MHC class II (tumor-intrinsic or antigen-presenting cell mediated); (iv) Checkpoint—Checkpoint blockade/Immune Modulation; (v) Infiltration—Increase immune infiltration into tumors; and (vi) Non-self—Promote tumor cell recognition as “non-self”. Selected genes of interest not in the above categories are colored black (‘Other’ category). ( e ) Gene Ontology (GO) Enrichment Analysis of the filtered top DE genes (FDR < 0.05 & |log2FC| > 0.1) identified via differential expression analysis between TRAF3 + and TRAF3 -cancer epithelial (CE) cells. X-axis represents the Fold Enrichment, and y-axis represents the immune-related Biological Process and Molecular Function GO terms, grouped into clusters based on functional similarity (for the full GO term graph with all the immune and non-immune related GO terms, see ). Dot size is analogous to the number of specific genes associated with each GO term, while their color gradient corresponds to the FDR-adjusted p -value (Q value). Abbreviations used include the following: CE (Cancer Epithelial cells), NE (Normal Epithelial cells), PVL (PeriVascular-Like cells), CAFs (Cancer-Associated Fibroblasts), PR (Positive Regulation), R (Regulation), prd (production), MM (Molecular Mediator), MBP (Macromolecule Biosynthetic Process), MMP (Macromolecule Metabolic Process), CR (Cellular Response), env/tal (environmental), RSP (receptor signaling pathway), SP (signaling pathway), resp. (response), ext. (external), and If-M (interferon-mediated).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Immunopeptidomics, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression in cancer cells affects PBMC subpopulations and cytokine expression. ( a ) FACs analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the downregulation of the CD25+CD127low (Tregs) subpopulation of CD4+ T cells. ( b ) FACS analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the upregulation of the CD56+CD16- subpopulation of NK-cells. ( c ) Diagrams depicting absolute quantification of IFN-γ, TNF-α and IL-10 in the supernatants of co-cultured PBMCs/MCF7-TRAF3 cells. ( d ) FACs analysis for live/dead MCF-7 breast cancer cells co-cultured with PBMCs depicting a shift from alive to dead cells in the MCF7-TRAF3 cell population in comparison to MCF7-control cells. ( e ) IHC stain for PD-L1 (CD274) on MCF7-control and MCF7-TRAF3. Arrowheads depict PD-L1 expression only on MCF7-control cells. ( f ) Schematic illustration of a proposed model of TRAF3 action in breast cancer epithelial cells and on the surrounding tumor microenvironmental cells.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Cell Culture, Quantitative Proteomics, Comparison, Control, Staining

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Immunofluorescence analysis (extended focus image) showing uromodulin on the surface of HEK293 cells. The protein is not assembled into polymers when expressed in this cellular system. Scale bar, 21 µm. ( B ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by MDCK and HEK293 cells. HEK293 cells only secrete the longer polymerisation-incompetent uromodulin isoform (black arrowhead), while MDCK cells also secrete the shorter one (white arrowhead). ( C ) Schematic representation of the selection process employed to identify candidate enzymes for the secretion of the short uromodulin isoform. Only membrane-bound serine proteases specifically expressed by MDCK cells and by the TAL segment of the nephron, but not by HEK293 cells, were selected. ( D ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by HEK293 cells expressing wild-type or catalytically inactive human hepsin or prostasin, as indicated. Only wild-type proteases promote the secretion of the short uromodulin isoform by these cells (white arrowhead). ( E ) Immunofluorescence analysis (extended focus image) showing uromodulin on the surface of HEK293 cells expressing wild-type or catalytically inactive hepsin or prostasin, as indicated. Uromodulin polymerisation is induced only when wild-type proteases are expressed. Scale bar, 21 µm. ( F ) Representative Western blot analysis of uromodulin immunoprecipitation (upper panels) from lysates of HEK293 cells expressing hepsin or prostasin, as indicated. Both enzymes are co-immunoprecipitated when uromodulin is co-expressed in HEK293 cells (lower panels). The arrowheads point at hepsin and prostasin specific bands. DOI: http://dx.doi.org/10.7554/eLife.08887.009

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Immunofluorescence, Western Blot, Selection, Membrane, Expressing, Immunoprecipitation

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) RT-PCR analysis showing gene expression of candidate proteases hepsin ( HPN ) and prostasin ( PRSS8 ) in MDCK and HEK293 cells. Constructs containing coding sequences of the human proteases were used as PCR positive controls (C+). Expression of GAPDH is shown as a cDNA positive control. PRSS8 and HPN are exclusively expressed in MDCK cells, confirming data obtained from available transcriptomes. ( B ) Representative Western blot analysis of uromodulin, hepsin and prostasin in lysates of transfected HEK293 cells. Wild-type proteases as well as catalytically inactive enzymes were expressed in HEK293 cells, as indicated. Protein disulfide-isomerase (Pdi) is shown as a loading control. The arrowhead points at hepsin specific band. DOI: http://dx.doi.org/10.7554/eLife.08887.010

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Construct, Expressing, Positive Control, Western Blot, Transfection, Control

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Schematic representation of human uromodulin domain structure as shown in . The region not included in recombinant efUmod is shadowed. ( B ) The deletion of the elastase-sensitive fragment of uromodulin does not affect protein polymerisation on the surface of MDCK cells, as shown by immunofluorescence analysis (efUmod wt). As for full-length uromodulin , this process depends on correct protein cleavage at the physiological site, since it is abolished when the consensus cleavage site is mutated (efUmod 4Ala, carrying the mutation 586 RFRS 589 > 586 AAAA 589 ). Scale bar, 50 µm. ( C ) Purified efUmod, either wild-type (efUmod wt) or mutated at the consensus cleavage site (efUmod YAla, carrying the mutation 586 RFRS 589 > 586 AYAA 589 ), was incubated with recombinant prostasin or hepsin, as indicated. Both proteases decrease the mass of wild-type efUmod (white arrowheads in upper and middle panels) and cause the loss of its C-terminal His-tag (lower panel). Hepsin is more efficient than prostasin, as it drives complete digestion of the product, despite being used at 20x lower concentration (picomolar ratio between protease and efUmod was 1:100 for hepsin and 1:5 for prostasin, see lanes 7 and 8 for comparison). The asterisk indicates His-tagged prostasin. DOI: http://dx.doi.org/10.7554/eLife.08887.011

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Recombinant, Immunofluorescence, Mutagenesis, Purification, Incubation, Concentration Assay, Comparison

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Confocal immunofluorescence analysis showing uromodulin (green), hepsin or prostasin (red) and E-cadherin (blue) (basolateral membrane marker) in polarised MDCK cells, as indicated. Upper panels represent the reconstruction on the xz axis of merged xy scans, for which a representative image is shown in lower panels. Both serine proteases co-localise with uromodulin on the apical plasma membrane of polarised MDCK cells. z stacks = 0.3 µm. A: apical, BL: basolateral. Scale bars, 5 µm. ( B ) Transcript levels of HPN and PRSS8 , as assessed by Real-Time qPCR in MDCK cells transfected with shRNA vectors, as indicated. Expression values (normalised to glyceraldehyde-3-phosphate dehydrogenase, GAPDH) are shown as relative to cells transfected with control vector. Expression of the proteases is specifically reduced in silenced cells. Bars indicate average ± s.e.m. **p<0.01, ***p<0.001 (Student’s t test). The graph represents mean ratios of 3 independent experiments . ( C ) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells transfected with control vector or with shRNA vectors targeting hepsin or prostasin, as indicated. Scale bar, 50 µm. Quantification of the average surface of uromodulin polymers shows that silencing of hepsin, but not of prostastin, substantially reduces uromodulin polymerisation on the membrane of MDCK cells. Bars indicate average ± s.e.m. ***p<0.001 (Mann-Whitney test). The graph represents mean ratios of 3 independent experiments . DOI: http://dx.doi.org/10.7554/eLife.08887.012 10.7554/eLife.08887.013 Figure 5—source data 1. Transcript level of HPN and PRSS8 in MDCK cells after shRNA transfection . DOI: http://dx.doi.org/10.7554/eLife.08887.013 10.7554/eLife.08887.014 Figure 5—source data 2. Quantification of the area of uromodulin polymers on the surface of MDCK cells after shRNA transfection . DOI: http://dx.doi.org/10.7554/eLife.08887.014

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Immunofluorescence, Membrane, Marker, Clinical Proteomics, Transfection, shRNA, Expressing, Control, Plasmid Preparation, MANN-WHITNEY

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Transcript level of Prss8 , as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh ). Expression of Prss8 is detected in proximal convoluted tubules (PCT), proximal straight tubules (PST) and, to a lesser extent, in thick ascending limb (TAL) and collecting ducts (CD). Bars indicate average ± s.e.m. of 3 independent experiments . ( B ) Immunofluorescence analysis of mouse kidney sections shows strong signal of endogenous prostasin on the apical plasma membrane of proximal tubules, and weak signal on the apical plasma membrane of TAL epithelial cells where it co-localises with uromodulin. Scale bar, 20 µm. ( C ) Representative Western blot analysis of urinary uromodulin from control Prss8 lox/lox or Prss8 -/- mice. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows that uromodulin secretion is comparable between Prss8 -/- mice and control Prss8 lox/lox animals (average ± s.d., n = 5/group, ) (Student’s t test). ( D ) Representative Western blot analysis of N-deglycosylated urinary uromodulin secreted by Prss8 -/- mice or control animals. An isoform of identical molecular weight, corresponding to the short uromodulin isoform, is detected in urine samples of both genotypes (n = 5/group). ( E ) Mass spectrometry sequence coverage (55% over the entire protein) of AspN-digested mouse uromodulin (UniProt accession Q91X17) purified from urine of Prss8 -/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at F588, the same C-terminal residue identified in urinary uromodulin of wild-type mice (Santambrogio et al., 2008) and control Prss8 lox/lox animals (data not shown). ( F ) Representative MS/MS spectrum confirming the sequence of urinary uromodulin C-terminal peptide ( 573 DSTSEQCKPTCSGTRF 588 ) in Prss8 -/- mice and table of fragmented ions. DOI: http://dx.doi.org/10.7554/eLife.08887.024

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Western Blot, Control, Concentration Assay, Molecular Weight, Mass Spectrometry, Sequencing, Purification, Residue, Tandem Mass Spectroscopy