Journal: The Journal of biological chemistry

Article Title: The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity.

doi: 10.1016/j.jbc.2023.103048

Figure Lengend Snippet: Figure 2. Inhibition of ADAMTS1 by TIMPs. A, inhibition of versicanase activity by TIMP family members. TIMP1, TIMP2, TIMP3, and TIMP4 (each at 500 nM) were incubated with ADAMTS1 (100 nM) for 1 h at 37 C before addition of V1-5GAG and digestion for 2 h. Following SDS-PAGE under reducing conditions (5% β-mercaptoethanol) and immunoblotting, FL V1-5GAG and versikine (VSK) were detected by the anti-Vc antibody. A representative immunoblot is shown (n = 2 independent experiments). B, inhibition of peptidolytic activity. TIMPs (each 25 nM) were incubated with a nominal con- centration of 25 nM ADAMTS1 for 1 h at 37 C before addition of the QF peptide substrate fluorescein-5(6)-carbonyl-Ala-Glu-Leu-Asn-Gly-Arg-Pro-Ile-Ser-Ile- Ala-Lys (5(6)-TAMRA) (3.5 μM) and digestion for 2 h. Following subtraction of the background (reactions not containing ADAMTS1), values were converted into percentage of ADAMTS1 activity in the absence of TIMPs and reported as average ± SD (n = 3, each point representing a technical replicate), p < 0.05 by Mann-Whitney test. C, titration of ADAMTS1 with TIMP3. TIMP3 (0–16 nM) was incubated with ADAMTS1 (20 nM nominal concentration) at 37 C for 1 h, and residual activity against the QF peptide FAM-AELNGRPISIAK-Tamra (3.5 μM) was determined. A representative titration curve is shown, each point representing a mean of two technical replicates. Final concentration of ADAMTS1 following titration was 10 nM. FL, full-length; No E, no enzyme; No I, no inhibitor; QF, Quenched-Fluorescent; TIMP, tissue inhibitor of metalloproteinase.

Article Snippet: Semiquantitative proteoglycan cleavage assays Purified V1-5GAG (100 nM) was digested with ADAMTS1, in TNC-B buffer at 37 C for 2 h. Where indicated, 500 μM recombinant human TIMP1, TIMP2, TIMP3, or TIMP4 (R&D Systems, Cat. n.: 970-TM, 971-TM, 973-TM, 974-TSF) were preincubated with 100 nM ADAMTS1 for 1 h at 37 C before digestion.

Techniques: Inhibition, Activity Assay, Incubation, SDS Page, Western Blot, MANN-WHITNEY, Titration, Concentration Assay

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: TIMP1 is overexpressed in CRC and correlates with poor prognosis. ( A ) Venn diagram showing the overlap of upregulated genes from four GEO datasets ( GSE127069 , GSE137511 , GSE54986 , and GSE14297 ), comparing normal tissues, primary CRC (pCRC), and colorectal liver metastases (CRLM). TIMP1, among other genes (CLDN1, IFITM2, SERPINE1), was identified as survival-related in the TCGA cohort. ( B ) Kaplan–Meier survival curves depicting overall survival in CRC patients from the TCGA cohort, stratified by TIMP1 expression levels (high vs. low). Median survival time is indicated. ( C ) Violin plots illustrating TIMP1 expression levels in normal tissues and CRC samples from the TCGA database. The left panel shows data for colon adenocarcinoma (COAD), and the right panel for rectal adenocarcinoma (READ). Statistical significance is indicated. ( D ) t-SNE plots derived from the GSE179784 dataset are shown, with cell type annotation presented in the left panel and TIMP1 expression patterns across different cell populations shown in the right panel. ( E ) A circular bar chart summarizes TIMP1 gene expression across a panel of colorectal cancer cell lines. Each bar corresponds to one cell line, and bar height reflects the relative expression level of TIMP1. ( F , G ) Quantification of TIMP1 mRNA expression in CRC tissues ( n = 20) and TIMP1 protein levels in serum samples ( n = 40). ( H ) Western blot showing TIMP1 protein levels in paired CRC tissues (T) and adjacent normal tissues (N). ( I , J ) Representative IHC images of TIMP1 expression in CRC and normal tissues (upper panels), with magnified views (lower panels) and corresponding IHC score comparison ( n = 110). Scale bars: 50 µm. ( K ) Kaplan–Meier survival analysis of CRC patients stratified by TIMP1 IHC score. p value was calculated by Log-rank test. Data are presented as mean ± SEM. Statistical significance was assessed by unpaired two-tailed Student’s t -test (two groups) or one-way ANOVA with post hoc tests (≥3 groups) and by log-rank test for survival. *** p < 0.001.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Expressing, Derivative Assay, Gene Expression, Western Blot, Comparison, Two Tailed Test

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: CRC-derived TIMP1 is secreted and primes the liver to promote metastasis. ( A ) Immunoblot of TIMP1 in conditioned media (CM) from MC38 and CT26 cells under control/vehicle or pLVX-TIMP1 overexpression. GAPDH, loading control of cell lysates. ( B ) ELISA quantification of TIMP1 in CM from MC38 and CT26 cells showing increased secretion upon oeTIMP1 and decreased secretion with two independent shRNA (sh1, sh2). ( C ) Liver-priming scheme in C57BL/6 mice: one intravenous dose of CM on day 1, day 7 intrasplenic injection of MC38-WT, and day 28 IVIS imaging and liver harvest. Representative IVIS images with quantification of normalized liver luminescence. Dose: protein-matched across groups; 100 μg total CM protein in 100 μL per mouse; TIMP1-CM 50 ± 8 ng/dose by ELISA; endotoxin < 0.1 EU/mL; vector-CM protein-matched; PBS vehicle. n = 5 mice/group. ( D ) Representative gross liver photographs and H&E staining (dashed outlines indicate metastatic areas) with liver-weight quantification for groups in (C). ( E ) Schematic of orthotopic priming: cecal implantation of MC38 vehicle or pLVX-TIMP1 cells, followed by intrasplenic injection of MC38-WT; representative IVIS liver images and quantification of normalized luminescence. n = 5 mice/group. ( F ) Corresponding gross/H&E liver images with quantification of liver weight and the percentage of liver metastases for groups in (E). ( G ) Kaplan–Meier overall-survival curves comparing vehicle and pLVX-TIMP1 groups. ( H ) Effects of TIMP1 knockdown (shTIMP1-1/-2) in MC38 cells on hepatic colonization: representative IVIS images and quantification of normalized liver luminescence. n = 5 mice/group. ( I ) Gross and H&E liver images with liver-weight and metastatic-percentage quantification for the knockdown cohorts in (H). ( J ) Kaplan–Meier overall survival analysis for vehicle and shTIMP1 groups. Data are presented as mean ± SEM. Statistical significance was assessed by unpaired two-tailed Student’s t -test (two groups) or one-way ANOVA with post hoc tests (≥3 groups) and by log-rank test for survival. * p < 0.05, *** p < 0.001.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Western Blot, Control, Over Expression, Enzyme-linked Immunosorbent Assay, shRNA, Injection, Imaging, Plasmid Preparation, Staining, Knockdown, Two Tailed Test

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: TIMP1 promotes CRLM in a macrophage-dependent manner. ( A ) Schematic diagram of the orthotopic CRC liver metastasis model. BALB/c nude mice were implanted with CT26 or MC38 cells transduced with pLVX-TIMP1 or vector control into the cecal wall, followed seven days later by intrasplenic injection of wild-type CRC cells to mimic hematogenous dissemination. ( B ) Representative in vivo bioluminescence (IVIS) images of the liver region and corresponding quantification of normalized liver luminescence in control versus TIMP1-overexpressing groups. n = 5 mice/group. ( C ) Experimental strategy for macrophage depletion: clodronate liposomes were administered i.p. starting on the day of cecal implantation (day 1) at 100 μL/mouse (~5 mg/kg clodronate equivalent), daily on days 1–6, then every 3 days thereafter until endpoint; PBS-loaded liposomes served as control. n = 5 mice/group. ( D ) Representative IVIS images and quantification of liver luminescence in control and TIMP1-overexpressing groups with or without clodronate liposome treatment. n = 5 mice/group. ( E ) Representative IVIS images and quantification of liver luminescence after neutrophil depletion with anti-Ly6G (clone 1A8): 200 μg/mouse i.p., starting on day 1, daily on days 1–6, then every 3 days thereafter until endpoint; depletion verified by flow cytometry. n = 5 mice/group. ( F ) Representative IVIS images and quantification of liver luminescence after NK-cell depletion with anti-NK1.1 (clone PK136): 200 μg/mouse i.p., starting on day 1, daily on days 1–6, then every 3 days thereafter until endpoint; depletion verified by flow cytometry. n = 5 mice/group. Data are shown as mean ± SEM, with each dot representing one animal. Statistical analysis was performed using one-way ANOVA followed by post hoc testing. *** p < 0.001; n.s., not significant. All IVIS images were acquired using identical exposure and filter settings, and quantification was performed on fixed-size regions of interest (ROIs) over the liver, background-subtracted and normalized for comparison across groups.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Transduction, Plasmid Preparation, Control, Injection, In Vivo, Liposomes, Flow Cytometry, Comparison

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: TIMP1 stimulation reprograms liver macrophage gene expression. ( A ) Schematic of the experimental design: Liver macrophages isolated from BALB/c mice were treated with recombinant TIMP1 (rTIMP1, 100 ng/mL) for 48 h, followed by RNA sequencing. ( B ) Volcano plot showing differentially expressed genes between rTIMP1-treated and control macrophages (fold change > 1.5, adjusted p < 0.05). Red: upregulated genes ( n = 308); green: downregulated genes ( n = 230); gray: non-significant. Selected genes are labeled. ( C ) qPCR validation of representative genes in RAW264.7 and THP-1-derived macrophages treated with rTIMP1 or vehicle (PBS). Data represent mean ± SEM ( n = 3). *** p < 0.001, unpaired two-tailed Student’s t -test. ( D ) Heatmap showing hierarchical clustering of differentially expressed genes in rTIMP1-treated and control groups. ( E ) Gene Ontology (GO) enrichment analysis of upregulated (red) and downregulated (green) genes in the categories of molecular function, cellular component, and biological process. ( F ) KEGG pathway enrichment analysis of differentially expressed genes. Size of the bubbles represents gene count; color indicates adjusted p -value. Key pathways including PI3K-Akt, cytokine–cytokine receptor interaction, and JAK-STAT signaling are enriched in the rTIMP1-treated group.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Gene Expression, Isolation, Recombinant, RNA Sequencing, Control, Labeling, Biomarker Discovery, Derivative Assay, Two Tailed Test

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: TIMP1 drives macrophage M2 polarization and pre-metastatic niche formation. ( A ) Immunofluorescence staining of RAW264.7 and THP-1-derived macrophages treated with PBS, IL4/IL13 (10 ng/mL, 48 h), or recombinant TIMP1 (rTIMP1). Cells were stained for CD163 (red) and CD206 (green); nuclei were counterstained with DAPI (blue). Scale bars: 50 µm. ( B ) Western blot analysis of M2 markers (CD204, CD206, CD163) in RAW264.7 and THP-1-derived macrophages following treatment as indicated. GAPDH served as a loading control. ( C ) Flow cytometric analysis and quantification of iNOS-positive macrophages in RAW264.7 and THP-1-derived macrophages after PBS, IL4/IL13, or rTIMP1 treatment. ( D ) Flow cytometric analysis and quantification of CD206-positive macrophages in the same groups. ( E ) Schematic of the cecal orthotopic injection model in BALB/c nude mice. Multiplex immunohistochemistry of liver tissues showing F4/80 (white) and CD206 (red) co-expression. Quantification indicates increased CD206 + F4/80 + macrophages in TIMP1-overexpressing group compared with control. Scale bars: 100 µm. Data are presented as mean ± SEM, *** p < 0.001.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Derivative Assay, Recombinant, Western Blot, Control, Injection, Multiplex Assay, Immunohistochemistry, Expressing

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: TIMP1 binds to CD63/β1-integrin on macrophages and activates the AKT/mTOR pathway. ( A ) Co-immunoprecipitation (Co-IP) showing the interaction between TIMP1 and CD63/β1-integrin in RAW264.7 and THP-1-derived macrophages. Cell lysates were immunoprecipitated with anti-TIMP1 or control IgG and immunoblotted for CD63, β1-integrin, and TIMP1. ( B ) Reverse Co-IP using anti-β1-integrin confirms the formation of a TIMP1-CD63-β1-integrin complex. ( C , D ) Flow cytometric analysis of CD163 + macrophages after treatment with PBS, rTIMP1, rTIMP1 + siCD63, or rTIMP1 + si-β1-integrin. Quantification of CD163 + cell percentages is shown (right). ( E ) Western blot analysis of p-AKT, p-PRAS40, and p-mTOR in macrophages treated with PBS, rTIMP1, or rTIMP1 + IL4/IL13. ( F ) Knockdown of CD63 or β1-integrin inhibits TIMP1-induced activation of the AKT/mTOR signaling pathway. GAPDH was used as a loading control. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance: *** p < 0.001.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Control, Western Blot, Knockdown, Activation Assay

Journal: Cells

Article Title: Integrin-Mediated TIMP1 Signaling Reprograms Liver Macrophages and Accelerates Colorectal Cancer Metastasis

doi: 10.3390/cells15010029

Figure Lengend Snippet: Cilengitide blocks the TIMP1/CD63/β1-integrin/mTOR axis and inhibits M2-macrophage-mediated CRLM. ( A ) Flow cytometry of CD163 + macrophages in RAW264.7 and THP-1–derived macrophages treated with PBS, recombinant TIMP1 (rTIMP1 100 ng/mL), or rTIMP1 plus Cilengitide 5 μM for 24 h; quantification on the right. ( B ) CD206 + macrophages measured as in (A) under the same treatments and timing. ( C ) Immunoblot of p-/total AKT, PRAS40, and mTOR in RAW264.7 and THP-1–derived macrophages after 24 h of PBS, rTIMP1 100 ng/mL, or rTIMP1 + Cilengitide 5 μM; GAPDH, loading control. ( D ) Orthotopic model in BALB/c nude mice with CT26: representative IVIS images and quantification of normalized liver luminescence showing enhanced metastatic burden with pLVX-TIMP1 and its suppression by Cilengitide. Cilengitide 5 mg/kg, i.p., in 200 μL every 3 days from day −1 to endpoint. ( E ) Corresponding whole-liver photos and H&E images (dashed lines, metastatic areas) with liver-weight quantification for the groups in (D). ( F ) Kaplan–Meier overall survival for vehicle, pLVX-TIMP1, and pLVX-TIMP1 + Cilengitide. ( G ) Validation using TIMP1-conditioned medium (TIMP1-CM) with CT26: representative liver IVIS images and quantification for PBS, TIMP1-CM, and TIMP1-CM + Cilengitide. One intravenous dose of CM on day 1, protein-matched across groups, 100 μg total protein in 100 μL per mouse; TIMP1 50 ± 8 ng per dose by ELISA; PBS as vehicle. Cilengitide 5 mg/kg, i.p., every 3 days starting day −1. ( H ) Gross liver and H&E images with liver-weight quantification for the groups in (G). ( I ) Kaplan–Meier overall survival for PBS, TIMP1-CM, and TIMP1-CM + Cilengitide. Data represent mean ± SEM. Statistical analysis: one-way ANOVA or log-rank test; * p < 0.05, *** p < 0.001.

Article Snippet: TIMP1 concentrations were quantified using a TIMP1 ELISA Kit (Proteintech, KE00833, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Derivative Assay, Recombinant, Western Blot, Control, Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

doi: 10.3390/ijms160613141

Figure Lengend Snippet: MMP and TIMP mRNA expression of all samples. Quantitative Real-Time PCR (qRT-PCR) analysis from torn RC tendons. The data represent the relative gene expression with 18S as reference gene using the Δ C t method with efficiency correction. Data are expressed as the mean ± SD ( n = 30) given in logarithmic form. MMP-2, TIMP-1, -2, and -3 showed the highest expression levels, while MMP-9, -10, and -13 showed lowest expression.

Article Snippet: TIMP-1 concentration was further analyzed by conventional sandwich ELISA (TIMP-1 DuoSet ELISA, R&D Systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

doi: 10.3390/ijms160613141

Figure Lengend Snippet: MMP and TIMP protein secretion of all samples. MMP-2 and TIMP-1 data were derived from sandwich ELISA. All other proteins were analyzed using Multiplex ELISA technique. All values were normalized to the total protein content (Coomassie Plus assay), given as mean ± SD ( n = 30) represented in a logarithmic graph. MMP-2, TIMP-1, and -2 protein secretion was strongest in the cells, while MMP-1, -3, and TIMP-4 protein secretion was lower.

Article Snippet: TIMP-1 concentration was further analyzed by conventional sandwich ELISA (TIMP-1 DuoSet ELISA, R&D Systems).

Techniques: Derivative Assay, Sandwich ELISA, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

doi: 10.3390/ijms160613141

Figure Lengend Snippet: MMPs and TIMPs grouped according to the age of the donors (under 65 years ( n = 16) and over 65 years ( n = 14)). ( A ) qRT-PCR was performed to analyze gene expression. The box plot data represent the relative gene expression with 18S as reference gene using the Δ C t method with efficiency correction. The mRNA levels of MMP-2, -9, -13 and TIMP-2, -3 are significantly increased with higher age; ( B ) Protein levels were analyzed using ELISA and normalized to total protein content (Coomassie Plus assay). Protein levels of MMP-2 and TIMP-1 were significantly elevated with higher age. To allow visualization of all MMPs/TIMPs in one figure, MMP-9 and -13 values were multiplied by 10 5 .

Article Snippet: TIMP-1 concentration was further analyzed by conventional sandwich ELISA (TIMP-1 DuoSet ELISA, R&D Systems).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

doi: 10.3390/ijms160613141

Figure Lengend Snippet: MMPs and TIMPs grouped according to the tear size (small tear size: Bateman score 1–2 ( n = 12); big tear size: Bateman score 3–4 ( n = 18)). ( A ) qRT-PCR was performed to analyze gene expression. The box plot data represent the relative gene expression with 18S as reference gene using the Δ C t method with efficiency correction. MMP-9, and -13 expression of donors with bigger tear size was significantly increased, while MMP-10 expression was decreased at mRNA level; and ( B ) Protein levels were analyzed using ELISA and normalized to total protein content (Coomassie Plus assay). Protein level of MMP-1 and TIMP-1 showed an elevated secretion with bigger tear size. To allow a visualization of all MMPs/TIMPs in one figure, MMP-1 values were multiplied by 10 2 and MMP-9, -10 and -13 by 10 5 .

Article Snippet: TIMP-1 concentration was further analyzed by conventional sandwich ELISA (TIMP-1 DuoSet ELISA, R&D Systems).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay