Journal: The European respiratory journal

Article Title: Hyperbaric oxygenation upregulates rat lung Na,K-ATPase.

doi: 10.1183/09031936.96.09030472

Figure Lengend Snippet: Fig. 6. – a) Effects of single hyperbaric oxygen (HBO) exposure on rat lung jun steady-state mRNA transcript levels. Relative steady state are shown for jun mRNA transcript level in arbitrary density units (a.u.) normalized to the 18S rRNA of the same samples are shown for control rats (not exposed to HBO), and rats after a single 2.5 h HBO exposure at different times post exposure. Data are presented as mean±SEM. : Control; : HBO. b) A representative auto- radiogram of rat lung jun steady-state mRNA. C: control. For abbre- viations see legend to figure 2.

Article Snippet: The autoradiograms were quantified by density scanning (Hoeffer Scientific GS300, San Francisco, CA, USA). α-1 Na,KATPase mRNA transcript level was normalized to the 18S rRNA of the same sample (American Type Culture Collection, Rockville, MD, USA).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A and B ) Primary stricturing CD patient-derived intestinal fibroblasts (CD-HIF) were pretreated with or without 100 μg/mL stricturing Crohn’s disease patients’ serum exosomes (CDSE). Two hours later, elafin was added and further incubated for 24 hours. n = 4 patients; 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( C ) Fresh human colonic tissues from 4 colon cancer patients were incubated in serum-free RPMI1640 medium with or without 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. n = 6 patients. Ordinary one-way ANOVA with Tukey test. ( D–F ) Primary stricturing CD-HIF were pretreated with or without 100 μg/mL CDSE. Two hours later, elafin was added and further incubated for 24 hours. ProCOL1A1, COL1A2, and fibronectin in cell lysates and hydroxyproline and C-telopeptide of type I collagen/ICTP in conditioned media were determined by ELISA. n = 4 patients; 4 independent experiments. Ordinary one-way ANOVA with Tukey test.

Article Snippet: Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A, left panel ) Serum-starved CD-HIF were treated with 100 μg/mL CDSE for 2 hours. Serum-starved CCD-18Co colonic fibroblasts were treated with 10 ng/mL TGF-β1 for 2 hours. Conditioned media were loaded to Proteome Profiler Human Protease Arrays (ARY021B; R&D Systems). A Bio-Rad ChemiDoc Imaging system captured the images. The rectangles highlighted the cathepsin S expression. ( A, right panel ) Quantification of cathepsin S signals (B7-8) and control signals (A1-2 and E1-2) using Bio-Rad Image Lab Software. Results were pooled from 3 independent experiments. Student t test was used to compare no serum exosome and CDSE groups. ( B, left panel ) Colonic cathepsin S mRNA expression in 40 non-IBD, 52 UC, 28 non-stricturing CD, and 15 stricturing CD patients was determined by real-time reverse transcription polymerase chain reaction. Ordinary one-way ANOVA test did not find any significant differences. ( B, middle panel ) Colonic cathepsin S mRNA (CTSS) expression in 43 CD patients is not correlated with colonic elafin mRNA expression. ( B, right panel ) Fresh human colonic tissues from 4 colon cancer patients were incubated in serum-free RPMI1640 media with or without 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. Ordinary one-way ANOVA test did not find any significant differences. ( C ) Cathepsin S activity assay was performed by incubating 2 μL of CS substrate (200 μmol/L final concentration), 94 μL CS reaction buffer, 2 μL cathepsin S inhibitor provided by the assay kit, 1 μL cathepsin S (0.4 μg/mL final concentration), and 1 μL elafin (0.5–10 μg/mL final concentration) at 37 o C for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). Results were pooled from 3 independent experiments. Ordinary one-way ANOVA with Tukey test. ( D ) Fresh human colonic tissues were pretreated with 100 μg/mL CDSE for 2 hours, followed by elafin 1 μg/mL for 2 hours. Conditioned media were collected. Each piece of tissue was homogenized in 500 μL CS cell lysis buffer. Next, 50 μg of tissue lysate supernatants in 50 μL CS lysis buffer or 50 μL of conditioned media were mixed with 2 μL CS substrates (200 μmol/L final concentration) and 48 μL CS reaction buffer and incubated for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). n = 6 patients. Ordinary one-way ANOVA with Tukey test. ( E ) CD-HIF in 96-well plates were pretreated with 100 μg/mL CDSE for 2 hours, followed by elafin 1 μg/mL for 2 hours. Conditioned media were collected. Cells were then lysed in 200 μL/well CS cell lysis buffer. Next, 50 μg of cell lysates in 50 μL CS lysis buffer or 50 μL of conditioned media were mixed with 2 μL of CS substrate (200 μmol/L final concentration) and 48 μL CS reaction buffer and incubated at 37 o C for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). Results were pooled from 6 independent experiments. Ordinary one-way ANOVA with Tukey test. ( F ) Serum-starved CD-HIF were pretreated with either 0.1% TFA r 0.4 μg/mL cathepsin S (1183-CY-010; R&D Systems) for 30 minutes, followed by 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. ProCOL1A1 protein was determined by ELISA. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test.

Article Snippet: Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems).

Techniques: Imaging, Expressing, Control, Software, Reverse Transcription, Polymerase Chain Reaction, Incubation, Activity Assay, Concentration Assay, Fluorescence, Lysis, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A and B ) Serum-starved CD-HIF were pretreated with 0.8% DMSO, 10 μmol/L PAR2 inhibitor GB88 (HY-120261; MCE), 10 μmol/L PAR1 agonist TRAP-6 (HY-P0078; MCE), or 10 μmol/L PAR2 agonist SLIGKV-NH 2 (HY-P0283; MCE). An hour later, the fibroblasts were exposed to 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test. ( C ) The most differentially expressed genes found in whole-transcriptome RNA sequencing in the colonic tissues of 2 stricturing and 2 non-stricturing CD patients. ( D ) STRING database analysis shows protein interaction association between the most differentially expressed genes in stricturing versus non-stricturing CD patients. ( E, upper panel ) Colonic ZEB1 mRNA expression in 40 non-IBD, 52 UC, 28 non-stricturing CD, and 15 stricturing CD patients. Stricturing CD patients have significantly higher colonic ZEB1 mRNA expression than non-stricturing CD patients. Ordinary one-way ANOVA with Tukey test. ( E, lower panel ) Positive correlation between colonic ZEB1 mRNA and collagen (ProCOL1A1) protein expression in 43 CD patients. ( F ) Serum-starved CD-HIF were transfected with either control (sc-37007; Santa Cruz Biotechnology, Dallas, TX) or ZEB1 (sc-38643; Santa Cruz Biotechnology) siRNA via lipofectamine 3000 overnight, followed by 100 μg/mL CDSE for 24 hours. ZEB1 and ProCOL1A1 proteins were measured by ELISA. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test for left panel. Student t test was used to compare ZEB1 expression between control siRNA and ZEB1 siRNA groups on right panel.

Article Snippet: Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems).

Techniques: Incubation, RNA Sequencing, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Serum-starved CD-HIF were transfected with either control or ZEB1-overexpressing construct via lipofectamine 3000 overnight. Fibroblasts were then pretreated with 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( B ) Efficiency of ZEB1-overexpressing construct transfection was determined by ELISA. Results were pooled from 4 experiments. Student t test was used to compare ZEB1 expression between control construct and ZEB1-overexpressing construct groups. ( C ) Serum-starved CD-HIF were pretreated with DMSO, 0.4 μg/mL cathepsin S, 10 μmol/L PAR1 agonist TRAP-6 (HY-P0078; MCE), or 10 μmol/L PAR2 agonist (HY-P0283; MCE) for 60 minutes, followed by addition of 100 μg/mL CDSE. Two hours later, some groups were treated with elafin (1 μg/mL) and incubated for 24 hours. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( D ) Fresh human colonic tissues from 4 colon cancer patients were incubated in serum-free RPMI1640 media with or without 100 μg/mL CDSE. Two hours later, elafin was added and further incubated for 24 hours. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( E ) Fresh human ileal tissues from 3 stricturing CD patients were incubated in serum-free RPMI1640 media with or without 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. COL1A1, COL1A2, and ZEB1 mRNA expression were determined by real-time reverse transcription polymerase chain reaction. Ordinary one-way ANOVA with Tukey test. ( F ) Macroscopic and microscopic morphology of the fresh stricturing ileal tissue. Intense collagen deposition is found in the mucosal layer.

Article Snippet: Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems).

Techniques: Transfection, Control, Construct, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Experimental plan. The formulation was suspended in mildly acidified (pH 5) water containing 0.5% hydroxypropyl methylcellulose (HPMC) and administered to TNBS-treated mice via oral gavage. The Eudragit polymer releases its drug in the mid-distal ileum and colon under an alkaline environment at ∼pH 8. ( B ) Elafin-Eudragit-HPMC (10 mg/kg) was administered to normal mice via oral gavage. Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems). Mean ± standard deviation. ( C ) H&E staining ( upper panels ) and MT staining ( lower panels ) of colonic tissues. Blue color in MT staining ( arrows ) indicated collagen deposition. ( D ) Histology scores, fibrosis scores, overall disease activities, colonic Zeb1 mRNA expression, and changes in body weight. Six mice per group. Ordinary one-way ANOVA with Tukey tests. ( E ) Comparison of intestinal tissue elafin levels and efficiencies in regulating target genes and disease activities. Body weight changes in elafin treatment groups compared with their respective positive control groups. Mean ± standard deviation.

Article Snippet: Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems).

Techniques: Formulation, Polymer, Enzyme-linked Immunosorbent Assay, Standard Deviation, Staining, Expressing, Comparison, Positive Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Serum-starved CCD-18Co fibroblasts were treated with DMSO, PAR1 inhibitor (SCH79797), or PAR2 inhibitor (GB88) for 2 hours. PAR2 inhibitor GB88 inhibited ERK1/2 phosphorylation. Results were pooled from 3 independent experiments. Ordinary one-way ANOVA with Tukey test. ( B ) Serum-starved CCD-18Co fibroblasts were pretreated with DMSO, PAR1 agonist (10 μmol/L), PAR2 agonists (10 μmol/L), or cathepsin S (0.4 μg/mL) for 30 minutes, followed by incubation with elafin (1 μg/mL) for 2 hours. ERK1/2 phosphorylation was determined by ELISA. PAR2 agonist and cathepsin S reversed elafin-mediated inhibition of ERK phosphorylation. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test.

Article Snippet: Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems).

Techniques: Phospho-proteomics, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Cancer cell

Article Title: Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.

doi: 10.1016/s1535-6108(04)00079-0

Figure Lengend Snippet: Figure 1. Homing specificity of metadherin phage

Article Snippet: A recent gene expression profiling study on breast cancer Experimental procedurespatients revealed that metadherin is overexpressed in many metastatic breast cancers. van ’t Veer et al. (2002) found that Cell lines, mice, and tumors metadherin mRNA expression levels (described as GenBank 4T1, a cell line derived from a Balb/c breast adenocarcinoma, was obtained entry AK000745) in breast cancer patients were significantly from ATCC and maintained as described by Pulaski and Ostrand-Rosenberg correlated with a poor prognosis due to metastasis.

Techniques:

Journal: Cancer cell

Article Title: Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.

doi: 10.1016/s1535-6108(04)00079-0

Figure Lengend Snippet: Figure 3. Lung-homing domain of metadherin is extracellular

Article Snippet: A recent gene expression profiling study on breast cancer Experimental procedurespatients revealed that metadherin is overexpressed in many metastatic breast cancers. van ’t Veer et al. (2002) found that Cell lines, mice, and tumors metadherin mRNA expression levels (described as GenBank 4T1, a cell line derived from a Balb/c breast adenocarcinoma, was obtained entry AK000745) in breast cancer patients were significantly from ATCC and maintained as described by Pulaski and Ostrand-Rosenberg correlated with a poor prognosis due to metastasis.

Techniques:

Journal: Cancer cell

Article Title: Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.

doi: 10.1016/s1535-6108(04)00079-0

Figure Lengend Snippet: Figure 4. Metadherin expression in 4T1 tumor cells

Article Snippet: A recent gene expression profiling study on breast cancer Experimental procedurespatients revealed that metadherin is overexpressed in many metastatic breast cancers. van ’t Veer et al. (2002) found that Cell lines, mice, and tumors metadherin mRNA expression levels (described as GenBank 4T1, a cell line derived from a Balb/c breast adenocarcinoma, was obtained entry AK000745) in breast cancer patients were significantly from ATCC and maintained as described by Pulaski and Ostrand-Rosenberg correlated with a poor prognosis due to metastasis.

Techniques: Expressing

Journal: Cancer cell

Article Title: Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.

doi: 10.1016/s1535-6108(04)00079-0

Figure Lengend Snippet: Figure 7. Anti-metadherin antibodies inhibit lung metastasis

Article Snippet: A recent gene expression profiling study on breast cancer Experimental procedurespatients revealed that metadherin is overexpressed in many metastatic breast cancers. van ’t Veer et al. (2002) found that Cell lines, mice, and tumors metadherin mRNA expression levels (described as GenBank 4T1, a cell line derived from a Balb/c breast adenocarcinoma, was obtained entry AK000745) in breast cancer patients were significantly from ATCC and maintained as described by Pulaski and Ostrand-Rosenberg correlated with a poor prognosis due to metastasis.

Techniques:

Journal: Cancer cell

Article Title: Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.

doi: 10.1016/s1535-6108(04)00079-0

Figure Lengend Snippet: Figure 8. siRNA reactive to metadherin mRNA inhibits lung metastasis

Article Snippet: A recent gene expression profiling study on breast cancer Experimental procedurespatients revealed that metadherin is overexpressed in many metastatic breast cancers. van ’t Veer et al. (2002) found that Cell lines, mice, and tumors metadherin mRNA expression levels (described as GenBank 4T1, a cell line derived from a Balb/c breast adenocarcinoma, was obtained entry AK000745) in breast cancer patients were significantly from ATCC and maintained as described by Pulaski and Ostrand-Rosenberg correlated with a poor prognosis due to metastasis.

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

doi: 10.1038/s41525-017-0033-4

Figure Lengend Snippet: Detection of a 548-nucleotide deletion in a HCoV 229E strain associated with myeloablation. a Depiction of the case history surrounding myeloablation and coronavirus infection. A woman in her 40s with AML was found to be infected with a HCoV 229E just before her HCT. Due to the patient’s advanced disease, the decision was made to continue with the transplant. Four successive HCoV-positive nasal swab specimens were available from the patient; two from before treatment with myeloablative cyclophosphamide and total body irradiation and two from after. All days are reported relative to HCT. b Coverage plots of mNGS reads mapped to the reference genome for HCoV 229E (NC_002645) of three samples from the patient revealed the generation of a 548-nucleotide deletion in the ORF4a/4b gene. In addition to deleting 83% of the coding region from ORF4a/4b, the deletion resulted in a frame shift that resulted in a premature stop codon 10 amino acids before the reference genome stop codon. Primer binding sites are denoted in blue for confirmatory junction RT-PCR. c Confirmatory junction RT-PCR of the four available HCoV-positive nasal swabs revealed the absence of the deletion before myeloablation and the presence of the deletion in specimens taken after myeloablation. NTC , no template control

Article Snippet: No CPE was visualized for any of the patient’s four clinical samples and HCoV 229E RNA levels in the culture supernatant were undetectable by qRT-PCR after inoculation, while viral growth was apparent in the positive control HCoV 229E ATCC type strain (Figure ).

Techniques: Infection, Irradiation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: NPJ Genomic Medicine

Article Title: Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

doi: 10.1038/s41525-017-0033-4

Figure Lengend Snippet: Specimens sequenced in this study

Article Snippet: No CPE was visualized for any of the patient’s four clinical samples and HCoV 229E RNA levels in the culture supernatant were undetectable by qRT-PCR after inoculation, while viral growth was apparent in the positive control HCoV 229E ATCC type strain (Figure ).

Techniques:

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: Adamts4 expression is increased in fibroblasts of septic mice. (A) Results of Reactome pathway enrichment analysis based on RNA‐seq data of mouse hearts after LPS stimulation ( n = 6). (B) Volcano plot of differentially expressed genes in LPS‐induced septic cardiomyopathy. Matrisome‐related genes are highlighted, with representative ECM‐remodelling genes, including Adamts4, Vcan, Timp1, Mmp14 , and Mmp9 , specifically labelled. (C) Representative Western blot images and statistical analysis of Adamts4 protein expression in mouse hearts 24 h after LPS treatment ( n = 6). (D) The level of Adamts4 mRNA relative to NS group in mouse hearts 24 h after LPS treatment ( n = 6). (E) Representative Western blot images and statistical analyses of the expression of ADAMTS4 protein in mouse fibroblasts following 24 h of LPS treatment ( n = 6). (F) The relative mRNA level of Adamts4 in mouse fibroblasts 24 h after LPS treatment ( n = 6). (G) Representative immunofluorescence images showing α‐SMA, ADAMTS4 and DAPI staining in heart sections from NS‐ and LPS‐treated mice. Arrowheads indicate ADAMTS4‐positive signals overlapping with or spatially associated with α‐SMA‐positive regions. Quantification shows the relative number of ADAMTS4 signals associated with α‐SMA‐positive regions. DAPI was used as a nuclear counterstain to show tissue cellularity. (H) Adamts4 mRNA levels in primary ECs, CMs, and CFs isolated from untreated wild‐type mouse hearts and subsequently stimulated with LPS in vitro for 24 h. (I) Reanalysis of the publicly available mouse cardiac single‐cell RNA‐seq dataset GSE207363 . Uniform Manifold Approximation and Projection (UMAP) plot showing the major cardiac cell populations identified from the public scRNA‐seq dataset GSE207363 . (J) Feature plot showing the distribution of Adamts4 expression across different cardiac cell populations in the scRNA‐seq dataset. (K) Comparison of Adamts4 ‐positive cell distribution between sham and septic cardiomyopathy samples, showing that Adamts4 expression is mainly enriched in fibroblast populations, with detectable changes in a subset of endothelial cells under septic conditions. Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. All tests were two‐sided. Two‐group comparisons were performed using an unpaired two‐tailed Student's t ‐test. For comparisons involving more than two groups (Figure ), one‐way ANOVA with Tukey's post hoc test was applied following Levene's test for homogeneity of variance; Welch's ANOVA with Tamhane's T2 correction was used when variances were unequal. Non‐normally distributed data were analysed using the Mann–Whitney U test or Kruskal–Wallis test with Dunn's correction, as appropriate. Statistical significance was set at p < .05.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Expressing, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Isolation, In Vitro, Single Cell, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: Tcf21‐lineage Adamts4‐deficient mice exhibit attenuated fibrosis and cardiac dysfunction in response to lipopolysaccharide (LPS) administration. (A) Tcf21‐lineage Adamts4‐deficient mice (Adamts4 cKO) were generated by crossing mice with conditional knockout alleles of Adamts4 (Adamts4 fl/fl ) with Tcf21‐iCreERT2 mice. (B) Representative Western blot images and statistical analysis of Adamts4 protein expression in CFs isolated from Adamts4 cKO mice and littermate controls ( n = 6). (C) Survival rate of mice challenged with LPS (15 mg/kg) ( n = 12). (D and E) Changes in ejection fraction (EF) and fractional shortening (FS) in different groups of mice after LPS stimulation ( n = 12). (F) Changes in left ventricular end‐systolic diameter (LVESD) after LPS stimulation in different groups of mice ( n = 8). (G and H) Changes in levels of LDH and CK‐MB in mouse serum after LPS stimulation in different groups of mice ( n = 6). (I) Representative echocardiograms of different groups of mice. (J) H&E staining. The black line indicates the thickness of myofibrils ( n = 6), and myofibrils become thinner after LPS stimulation. (K) PSR staining. The area of cardiac tissue fibrosis in Adamts4 cKO mice decreases after LPS stimulation ( n = 6). Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. All tests were two‐sided. For two‐group comparisons (Figure 2B), an unpaired two‐tailed Student's t ‐test was used. For multiple comparisons (Figure 2D–K), one‐way ANOVA with Tukey's post hoc test was applied following confirmation of variance homogeneity by Levene's test; Welch's ANOVA with Tamhane's T2 correction was used when variances were unequal. Survival analysis (Figure 2C) was performed using the Kaplan–Meier method with the log‐rank (Mantel–Cox) test. Non‐parametric alternatives (Mann–Whitney U test or Kruskal–Wallis test with Dunn's correction) were employed when data were non‐normally distributed. Analyses were performed using GraphPad Prism 10. A p value < .05 was considered statistically significant. * p < .05 versus littermate Adamts4 fl/fl controls; # p < .05 compared with the LPS‐stimulated Adamts4 cKO group.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Generated, Knock-Out, Western Blot, Expressing, Isolation, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: Tcf21‐lineage Adamts4‐deficient mice display attenuated myocardial inflammation in response to lipopolysaccharide (LPS) administration. (A) The expression levels of cytokines such as IL‐6, TNF‐α, IL‐1β, IL‐10, MCP1 and IL‐8 in samples from Adamts4 fl/fl mice (control group) and Adamts4 cKO mice were detected, and the right‐side scale shows the relative expression fold ( n = 5). (B–E) The protein expression of inflammation‐related indicators IKKβ, phosphorylated IKKβ (p‐IKKβ), IκBα, phosphorylated IκBα (p‐IκBα), p65, and phosphorylated p65 (p‐p65) was detected ( n = 6). (F–I) The protein expression levels of apoptosis‐related indicators Bax, Bcl‐2, Caspase‐3 and Cleaved‐Caspase‐3 were detected ( n = 6). (J) Representative images of DHE distribution detected by immunofluorescence, and the DHE fluorescence intensity was counted ( n = 6). (K) The TUNEL method was used to detect the apoptosis of cells in myocardial tissue sections of mice in different groups, and the cell apoptosis rate was calculated ( n = 6). Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. All tests were two‐sided. For two‐group comparisons (Figure 3B), an unpaired two‐tailed Student's t ‐test was used. For multiple comparisons (Figure 3A,C–K), one‐way ANOVA with Tukey's post hoc test was applied following confirmation of variance homogeneity by Levene's test; where variances were unequal, Welch's ANOVA with Tamhane's T2 correction was used. Non‐parametric tests (Mann–Whitney U test or Kruskal–Wallis test with Dunn's correction) were employed when data deviated from normality. Statistical significance was set at P < 0.05. * p < .05 versus littermate Adamts4 fl/fl controls; # p < .05 compared with the LPS‐stimulated Adamts4 cKO group.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Expressing, Control, Immunofluorescence, Fluorescence, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: Postn‐lineage Adamts4‐deficient mice exhibit attenuated LPS‐induced cardiac dysfunction and myocardial fibrosis. (A and B) By crossing Adamts4 knockout mice (Adamts4 fl/fl ) with Postn‐CreER mice, we successfully developed Postn‐lineage Adamts4‐deficient mice. The figure displays representative Western blot images and statistical analysis results of Adamts4 protein expression in cardiac fibroblasts (CFs) isolated from Adamts4 mfKO mice and littermate Adamts4 fl/fl controls ( n = 6). (C) Survival curves of mice challenged with LPS (15 mg/kg) ( n = 12). (D and E) Dynamic changes in ejection fraction (EF) and fractional shortening (FS) in different groups of mice after LPS stimulation ( n = 12). (F and G) Changes in serum levels of lactate dehydrogenase (LDH) and creatine kinase‐MB (CK‐MB) in different groups of mice after LPS stimulation ( n = 6). (H) Representative immunofluorescence images showing DHE distribution and quantification of DHE fluorescence intensity ( n = 6). (I) TUNEL staining and quantification of apoptotic cells in myocardial sections from different groups of mice ( n = 6). (J) PSR staining showing reduced cardiac fibrosis area in Adamts4 mfKO mice after LPS stimulation ( n = 6). Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. All tests were two‐sided. For two‐group comparisons (Figure 4B), an unpaired two‐tailed Student's t ‐test was used. For multiple comparisons (Figure 4D–J), one‐way ANOVA with Tukey's post‐hoc test was applied following Levene's test for homogeneity of variance; Welch's ANOVA with Tamhane's T2 correction was used when variances were unequal. Survival analysis (Figure 4C) was performed using the Kaplan–Meier method with the log‐rank (Mantel–Cox) test. Non‐normally distributed data were analysed using the Mann–Whitney U test or Kruskal–Wallis test with Dunn's correction. Statistical significance was set at p < .05. * p < .05 versus littermate Adamts4 fl/fl controls; # p < .05 compared with the LPS‐stimulated Adamts4 mfKO group.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Knock-Out, Western Blot, Expressing, Isolation, Immunofluorescence, Fluorescence, TUNEL Assay, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: Tcf21‐lineage fibroblast Adamts4 overexpression exacerbates lipopolysaccharide (LPS)‐induced myocardial fibrosis and cardiac dysfunction. (A and B) Conditional Tcf21‐lineage fibroblast Adamts4 overexpression mice (Adamts4 cTg) were generated by crossing Adamts4 Tg fl/fl conditional transgenic with Tcf21‐iCreERT2 mice. Representative Western blot images and statistical analysis of Adamts4 protein expression in cardiac fibroblasts (CFs) isolated from Adamts4 cTg mice and littermate controls ( n = 6). (C) Survival rate of mice challenged with LPS (15 mg/kg) ( n = 12). (D and E) Changes in ejection fraction (EF) and fractional shortening (FS) in different groups of mice after LPS stimulation ( n = 12). (F) Changes in left ventricular end‐systolic diameter (LVESD) in different groups of mice after LPS stimulation ( n = 8). (G and H) Changes in levels of LDH and CK‐MB in mouse serum in different groups of mice after LPS stimulation ( n = 6). (I) H&E staining: Black lines indicate the thickness of myofibrils ( n = 6). Myofibrils in Adamts4 cTg mice were thinner after LPS stimulation. (J) PSR staining: After LPS stimulation, the fibrosis area of cardiac tissue in Adamts4 cTg mice increased ( n = 6). (K) The expression levels of cytokines such as IL‐6, TNF‐α, IL‐1β, IL‐10, MCP1 and IL‐8 in samples from Adamts4 Tg fl/fl mice (control group) and Tcf21‐lineage fibroblast Adamts4 overexpression (cTg) mice were detected, and the right‐side scale shows the relative expression fold ( n = 5). (L) Representative immunofluorescence images showing DHE distribution and quantification of DHE fluorescence intensity in different groups of mice ( n = 6). Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. All tests were two‐sided. For two‐group comparisons (Figure 5B), an unpaired two‐tailed Student's t ‐test was used. For multiple comparisons (Figure 5D–L), one‐way ANOVA with Tukey's post‐hoc test was applied following Levene's test for homogeneity of variance; Welch's ANOVA with Tamhane's T2 correction was used when variances were unequal. Survival analysis (Figure 5C) was performed using the Kaplan–Meier method with the log‐rank (Mantel–Cox) test. Non‐normally distributed data were analysed using the Mann–Whitney U test or Kruskal–Wallis test with Dunn's correction. Statistical significance was set at p < .05. * p < .05 versus littermate Adamts4 Tg fl/fl controls; # p < .05 compared with the LPS‐stimulated Adamts4 cTg group.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Over Expression, Generated, Transgenic Assay, Western Blot, Expressing, Isolation, Staining, Control, Immunofluorescence, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: ADAMTS4 promotes cardiac fibroblast activation through direct interaction with and cleavage of TSP1. (A) Mouse cardiac fibroblasts (CFs) were transfected with shRNA targeting Adamts4 ( shAdamts4 ) or control shRNA and stimulated with LPS. Protein expression of α‐SMA was assessed by Western blotting and quantified relative to GAPDH. (B) CFs with Adamts4 knockdown were stimulated with LPS, and α‐SMA mRNA levels were measured by qPCR. (C) CFs were infected with lentivirus encoding full‐length Adamts4 ( oeAdamts4 ) or control vector and stimulated with LPS. Protein expression of α‐SMA was analysed by Western blotting and quantified relative to GAPDH. (D) CFs overexpressing Adamts4 were stimulated with LPS, and α‐SMA mRNA levels were measured by qPCR. (E) Co‐immunoprecipitation (Co‐IP) combined with LC‐MS analysis was performed using heart tissue lysates from LPS‐treated mice. Representative high‐confidence candidate proteins are listed, with thrombospondin‐1 (TSP1) identified as a potential interacting partner of ADAMTS4. (F) Reciprocal Co‐IP assays were performed in heart tissue lysates using antibodies against ADAMTS4 or TSP1, followed by immunoblotting to validate the interaction between ADAMTS4 and TSP1. (G) GST pull‐down assay was conducted to determine whether ADAMTS4 directly binds to TSP1. GST‐tagged ADAMTS4 and HIS‐tagged TSP1 were used, and bound proteins were detected by immunoblotting. (H) Time‐course analysis of ADAMTS4‐mediated TSP1 processing in CFs overexpressing Adamts4 following LPS stimulation. Cells and conditioned supernatants were collected at the indicated time points. Full‐length intracellular proteins were normalized to GAPDH, whereas secreted TSP1‐derived fragments in conditioned media were normalized to total membrane protein staining. (I) CFs isolated from Adamts4 cTg mice and their littermate controls were stimulated with LPS. Protein levels of ADAMTS4, full‐length TSP1, and TSP1 fragments were analysed by Western blotting and quantified. (J) CFs were subjected to Adamts4 overexpression with or without Tsp1 knockdown and stimulated with LPS. α‐SMA mRNA levels were measured by qPCR. (K) CFs were subjected to Adamts4 overexpression with or without Tsp1 knockdown and stimulated with LPS. Col1a1 mRNA levels were measured by qPCR. Data are presented as mean ± SEM. Normality and homogeneity of variance were assessed using Shapiro‐Wilk and Levene's tests, respectively. Two‐group comparisons were made using unpaired two‐tailed Student's t ‐test or Mann–Whitney U test, as appropriate. Multiple‐group comparisons were performed using one‐way ANOVA with Tukey's post hoc test (equal variances) or Tamhane's T2 test (unequal variances); non‐normally distributed data were analysed via Kruskal–Wallis test with Dunn's post‐hoc correction. All tests were two‐sided, with p < .05 considered significant. * p < .05 versus respective control group; # p < .05 versus the corresponding LPS‐treated control.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Activation Assay, Transfection, shRNA, Control, Expressing, Western Blot, Knockdown, Infection, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Pull Down Assay, Derivative Assay, Membrane, Staining, Isolation, Over Expression, Two Tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

doi: 10.1002/ctm2.70769

Figure Lengend Snippet: ADAMTS4‐mediated cleavage of TSP1 at residues 236–246 drives fibroblast activation via TGF‐β/Smad and NF‐κB signalling. (A) Schematic representation of full‐length mouse TSP1 highlighting predicted ADAMTS4 cleavage sites within the N‐terminal region. Two candidate cleavage regions (Site1 and Site2) identified by protease prediction analysis are indicated. (B) Alanine substitution strategy for candidate cleavage motifs within TSP1. Three regions selected based on prediction and structural accessibility (236–246 aa, 298–308 aa, and 322–332 aa) were mutated to alanine. (C) Sequence alignment of candidate cleavage regions across Homo sapiens , Mus musculus and Rattus norvegicus , demonstrating conservation of key residues. (D) Cleavage of TSP1 was assessed by Western blotting using antibodies against HA (full‐length and fragment), Flag (ADAMTS4), and GAPDH. (E) Full‐length intracellular proteins were normalized to GAPDH, whereas secreted TSP1‐derived fragments in conditioned media were normalized to total membrane protein staining. The resulting ratios were quantified and statistically analysed. (F) NIH/3T3 fibroblasts were transfected with wild‐type ADAMTS4 or the catalytically inactive ADAMTS4‐E362A mutant and subsequently stimulated with 10 µg/mL LPS for 48 h, with or without TIMP3 treatment. (G) NIH/3T3 fibroblasts subjected to shRNA‐mediated Tsp1 knockdown were reconstituted with HA‐tagged wild‐type TSP1 (WT‐TSP1) or the cleavage‐resistant Mut1‐TSP1 variant, co‐transfected with Flag‐tagged ADAMTS4, and subsequently stimulated with 10 µg/mL LPS for 48 h. Full‐length TSP1, ADAMTS4, α‐SMA and GAPDH in cell lysates, together with secreted TSP1‐derived fragments in concentrated conditioned media, were assessed by Western blotting. (H) Densitometric quantification of full‐length TSP1, secreted TSP1‐derived fragments, ADAMTS4, and α‐SMA from (G). Intracellular proteins were normalized to GAPDH, whereas secreted TSP1‐derived fragments were normalized to the total protein signal in the corresponding conditioned‐medium lane. (I) Western blot analysis of downstream signalling pathways in fibroblasts from (G), including phosphorylated and total Smad2 and NF‐κB p65. (J) Quantification of p‐Smad2/Smad2 and p‐p65/p65 ratios from (I). Data are presented as mean ± SEM. Normality and homogeneity of variance were assessed using Shapiro–Wilk and Levene's tests, respectively. Two‐group comparisons were performed using unpaired two‐tailed Student's t ‐test or Mann–Whitney U test as appropriate. Multiple‐group comparisons were conducted using one‐way ANOVA with Tukey's post hoc test or Tamhane's T2 test; non‐parametric data were analysed using Kruskal–Wallis test with Dunn's post hoc correction. All tests were two‐sided, with p < .05 considered statistically significant. * p < .05; ns, not significant.

Article Snippet: Serum and cardiac tissue ADAMTS4 levels were measured using a Mouse ADAMTS4 ELISA Kit (RK08749; ABclonal) according to the manufacturer's instructions.

Techniques: Activation Assay, Sequencing, Western Blot, Derivative Assay, Membrane, Staining, Transfection, Mutagenesis, shRNA, Knockdown, Variant Assay, Two Tailed Test, MANN-WHITNEY