Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 1. Immunohistochemistry and immunofluorescence of coronary thrombus. (A) Aspirated material in a patient with STEMI due to total occlusion of coronary artery. (B) H&E staining of coronary artery thrombus. (C and D) Immunohistochemistry of coronary thrombus with MMP12 antibody. Arrows indicate examples of MMP12 positive cells. (E and F) Immunofluorescence staining of coronary thrombus with MMP12 antibody. Chartreuse: MMP12, Blue: Counterstain nucleus with DAPI. (E) Negative control. (F) Experimental group (magnification 400). Analysis of staining from 3 independent experiments showed similar results. STEMI=ST- segment elevation myocardial infarction.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 3. The plasma levels of MMP12 and TIMP1 in STEMI, SAP, and control groups. (A) Columns and lines show significant increase in MMP12 concentration but lower activity in STEMI compared with SAP group and normal coronary artery control group. (B) The STEMI group had significantly higher TIMP1 concentration than SAP group and control group. (C) The imbalance in MMP/TIMP ratio was observed in STEMI group compared with SAP control group. The assays were performed in triplicate and the results expressed as the mean plus or minus SEM. ∗P<.05, means significant difference between 2 groups by Mann–Whitney test. SAP=stable angina pectoris, STEMI=ST-segment elevation myocardial infarction.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Clinical Proteomics, Control, Concentration Assay, Activity Assay, MANN-WHITNEY

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 2. MMP12 was present in human coronary thrombi. (A) RT-PCR analysis of MMP12 gene fragment. Lane 1: DNA ladder; lanes 2–6: coronary thrombus; GAPDH blot (bottom panel) was included as loading control. (B) WB analysis of MMP12 expression in coronary artery thrombus by SDS–PAGE. Lane 1: molecular weight markers; lanes 2–6: WB products from coronary artery thrombus of STEMI-LTB; lane 7: recombinant human MMP12 with molecular weights of 45kDa. (C) The caseinolytic activity of MMP12 in coronary thrombi. Lane 1: molecular weight markers; lanes 2–4: zymography products from the coronary artery thrombus of STEMI patients; lane 5: zymography products from the coronary artery thrombus of STEMI patients incubated with antihuman MMP12 antibody. Lane 6: Recombinant human MMP12 with molecular weights of 45kDa. Analysis of the blots from 3 independent experiments showed similar results. LTB=large thrombus burden, RT-PCR=reverse transcription polymerase chain reaction, STEMI=ST-segment elevation myocardial infarction, WB=Western blot.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, SDS Page, Molecular Weight, Recombinant, Activity Assay, Zymography, Incubation, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Medicine

Article Title: Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction

doi: 10.1097/md.0000000000008035

Figure Lengend Snippet: Figure 4. The plasma levels of MMP12 and TIMP1 in LTB and STB subgroups. (A) Columns and lines show significant increase in MMP12 concentration (P<.05) but lower activity (P>.05) in LTB compared with STB subgroup. (B) The LTB subgroup had significantly higher TIMP1 concentration than STB subgroup. (C) Although the difference did not reach statistical significance, the imbalance in MMP/TIMP ratio were higher in LTB than patients with STB. The assays were performed in triplicate and the results expressed as the mean plus or minus SEM. ∗P<.05, means significant difference between 2 groups by Mann–Whitney test. LTB=large thrombus burden, STB=small thrombus burden.

Article Snippet: After incubation with a primary antibody: Anti-MMP12 antibody (EP1261Y, rabbit monoclonal antibody, corresponding human MMP12 aa E450 to the C terminal, MilliporeSigma, Darmstadt, Germany) overnight at 4°C, a secondary antibody (goat antirabbit IgG-B; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 30minutes at 37°C.

Techniques: Clinical Proteomics, Concentration Assay, Activity Assay, MANN-WHITNEY

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Integrin β6 is a proteolysis‐resistant mediator of CRC tumour cell adhesion to endothelial cells. (A) Lysates of HT‐29 and DLD‐1 cells harvested in the presence of 0.05% trypsin or 5 mM EDTA‐PBS were collected, and the expression of integrins αv, β6, β1 and β5 was analysed using western blotting. Blots show the full‐length proteins as well as the trypsin‐induced protein fragments. GAPDH was used as a loading control. (B) Adhesion of cytopainter‐stained ITGB6 KO or NT cells harvested by trypsinization or 5 mM PBS‐EDTA to an IL‐1β‐stimulated HUVEC monolayer under static conditions. Arrow points towards an adherent cell. Scale bar: 250 μm. The quantification is shown below. Mann–Whitney U test; ns, not significant; ** p < 0.01. (C) The cancer‐related survival (60‐month cut‐off) of CRC patients who did not receive neoadjuvant therapy and had no residual tumour (R0) was explored. Based on MMP11 and MMP12 RNA expression levels, patients were divided into high‐ and low‐MMP groups. ITGB6 ‐dependent survival was investigated in these groups. The number of patients per group is indicated by ‘ n ’. Cox proportional hazards survival regression. (D) Recombinant integrins αvβ6, αvβ1, and αvβ5 were incubated with MMP‐12 at an enzyme‐to‐substrate ratio of 1:10 for 1 h. Proteolytic cleavage of integrins was evaluated via western blotting.

Article Snippet: MMP‐12 (R&D Systems, Catalogue #: 917‐MPB) was activated in a concentration of 50 ng/μL by incubation in TCN buffer (10 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5) at 37°C for 2 h. Following enzyme activation, the recombinant human integrins (integrin αvβ6; Catalogue #: T6‐H52E1, integrin αvβ1; Catalogue #: IT1‐H52E1, integrin αvβ5; Catalogue #: IT5‐H52W5, AcroBiosystems, Newark, DE, USA) were added in an enzyme‐to‐substrate ratio of 1:10 in TCN buffer for 1 h (MMP‐12) at 37°C.

Techniques: Expressing, Western Blot, Control, Staining, MANN-WHITNEY, RNA Expression, Recombinant, Incubation

Journal: BMB Reports

Article Title: SPINK1 promotes cell growth and metastasis of lung adenocarcinoma and acts as a novel prognostic biomarker

doi: 10.5483/bmbrep.2018.51.12.205

Figure Lengend Snippet: Fig. 3. SPINK1 enhances the migration and invasion of LAC cells by up-regulating MMP12. (A) MMPs expressions were analyzed by qRT-PCR in A549 cells and H1975 cells transfected with scramble or SPINK1 siRNAs. (B) MMP12 protein expressions were analyzed by Western blot in A549 cells and H1975 cells transfected with scramble or SPINK1 siRNA 1. (C) MMP12 protein levels were measured by Western blot in PC9 cells and H1299 cells transfected with control or SPINK1. (D) MMP12 mRNA expression in xenograft tissues was measured by qPCR. (E) The correlation between SPINK1 expression and MMP12 expression in LAC tissues was analyzed with Pearson correlation. (F) The interference effect of MMP12 siRNA was verified by Western blot in PC9 and H1299 cells stably overexpressing SPINK1. The number of migrating and invasive cells was counted (G). (H) Recombinant human MMP12 (rhMMP12) or control IgG (cIgG) at a concentration of 50 ng/ml was added back into SPINK1 knockdown A549 and H1299 cells, and the number of migrating and invasive cells was counted. *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001.

Article Snippet: Recombinant human MMP12 (rhMMP12, 917-MPB) and the control IgG (cIgG, 1-001-A) were purchased from R&D Systems.

Techniques: Migration, Quantitative RT-PCR, Transfection, Western Blot, Control, Expressing, Stable Transfection, Recombinant, Concentration Assay, Knockdown

Journal: Advanced Science

Article Title: Resident Macrophage‐Orchestrated Immune and Fibroblast Interactions in Immune Checkpoint Inhibitor‐Associated Nephrotoxicity

doi: 10.1002/advs.202505445

Figure Lengend Snippet: Transcriptomic and protein‐level evidence of MMP12 and CXCL9 upregulation in ICI‐associated nephrotoxicity (ICI‐AN). A) Volcano plot of differentially expressed genes in mouse kidney tissue after ICI treatment, highlighting significantly upregulated genes (e.g., MMP12, CXCL9, CXCL10, and CD74) in red. Differentially expressed genes were identified using a threshold of |log 2 (Fold Change)| > 1.5 and FDR < 0.05. Data were obtained from the GEO database. B) Biological process (BP) enrichment analysis for the upregulated genes in ICI‐AN. C) KEGG Pathway enrichment analysis. D) Immunohistochemical staining of MMP12 in human kidney tissue from control and ICI‐AN group. Scale bars: 50 µm; magnification: 400×. E) CXCL9 levels in urine ( n = 10 control, n = 9 AIN, n = 6 ICI‐AN) and serum ( n = 10 control, n = 10 AIN, n = 7 ICI‐AN). F) Correlation analysis between urinary or serum CXCL9 levels and resident macrophage frequency. Spearman's rank correlation coefficients (R) and P ‐values are shown. Data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA with Tukey's post hoc test. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Membranes were blocked with 5% non‐fat milk and incubated overnight at 4 °C with primary antibodies targeting MMP12 (Novus Biologicals, Centennial, CO, USA), α‐SMA (Proteintech, Wuhan, China), NGAL (R&D systems, USA), and β‐actin (Proteintech, Wuhan, China), followed by incubation with HRP‐conjugated secondary antibodies.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Advanced Science

Article Title: Resident Macrophage‐Orchestrated Immune and Fibroblast Interactions in Immune Checkpoint Inhibitor‐Associated Nephrotoxicity

doi: 10.1002/advs.202505445

Figure Lengend Snippet: Resident macrophages mediate regional enrichment of MMP12 and induce CXCL9 production. A) Western blot analysis of fibrosis and inflammatory markers. B) Quantitative polymerase chain reaction (qPCR) analysis of pro‐inflammatory and fibrosis‐related genes. C) Immunohistochemical staining of MMP12 in kidney tissue from mice treated with either anti‐IgG or anti‐PD‐1 antibody. Red arrowheads indicate MMP12‐positive cells. Scale bars: 50 µm; magnification: 400×. D) Kidney sections stained with DAPI (blue, nuclei), tdTomato (red, resident macrophages), and MMP12 (green). The merged image shows the colocalization of MMP12 with resident macrophages, highlighted in the magnified region (dashed box). TdTomato signal intensity may vary across figures due to differences in imaging settings and sample preparation. Scale bars: 100 µm; magnification: 630×. E) CXCL9 levels in mouse serum. F) qPCR results of CD8 + T cells co‐cultured with resident macrophages ( n = 3 per group). G) qPCR results of resident macrophages isolated from mouse kidneys treated with anti‐PD‐1 antibody or control IgG ( n = 3 per group). Control, n = 3; LLC, n = 3; LLC + anti‐IgG, n = 3; LLC + anti‐PD‐1, n = 5; Resident macrophage depleted + LLC + anti‐IgG, n = 3; Resident macrophage depleted + LLC + anti‐PD‐1, n = 5. Data are presented as mean ± SD. Statistical comparisons were made using an unpaired t ‐test or one‐way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Membranes were blocked with 5% non‐fat milk and incubated overnight at 4 °C with primary antibodies targeting MMP12 (Novus Biologicals, Centennial, CO, USA), α‐SMA (Proteintech, Wuhan, China), NGAL (R&D systems, USA), and β‐actin (Proteintech, Wuhan, China), followed by incubation with HRP‐conjugated secondary antibodies.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Imaging, Sample Prep, Cell Culture, Isolation, Control

Journal: Advanced Science

Article Title: Resident Macrophage‐Orchestrated Immune and Fibroblast Interactions in Immune Checkpoint Inhibitor‐Associated Nephrotoxicity

doi: 10.1002/advs.202505445

Figure Lengend Snippet: Pharmacological inhibition of MMP12 and neutralization of CXCL9 attenuate renal injury and immune activation in ICI‐associated nephrotoxicity (ICI‐AN). A) Schematic of the experimental timeline in mice treated with MMP408. B) Serum creatinine (Cr) levels in each treatment group ( n = 5 per group). C) Representative histological images of kidney tissues stained with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and Masson's trichrome. Red arrowheads indicate inflammatory cell infiltration; yellow arrowheads indicate tubular injury. Scale bars: 50 µm. D) Western blot analysis of NGAL and α‐SMA protein expression in renal tissues ( n = 5 per group). E) qPCR analysis of Ngal and Fn1 mRNA expression in kidney tissues ( n = 5 per group). F) qPCR analysis of fibrosis‐related genes ( Acta2 , Fn1 , and Tgfb1 ) in NRK‐49F fibroblasts after 48‐hour transwell co‐culture with resident macrophages isolated from anti‐PD‐1‐treated mice, with or without MMP408 pretreatment ( n = 3 per group). G) Flow cytometric analysis of CD25 mean fluorescence intensity (MFI) on CD8⁺ T cells co‐cultured with resident macrophages in the presence or absence of CXCL9 neutralizing antibody ( n = 3 per group). H) qPCR analysis of Gzmb and Il1b expression in CD8⁺ T cells after 48‐hour co‐culture with resident macrophages with or without α‐CXCL9 treatment ( n = 3 per group). Data are presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA or unpaired t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Membranes were blocked with 5% non‐fat milk and incubated overnight at 4 °C with primary antibodies targeting MMP12 (Novus Biologicals, Centennial, CO, USA), α‐SMA (Proteintech, Wuhan, China), NGAL (R&D systems, USA), and β‐actin (Proteintech, Wuhan, China), followed by incubation with HRP‐conjugated secondary antibodies.

Techniques: Inhibition, Neutralization, Activation Assay, Staining, Western Blot, Expressing, Co-Culture Assay, Isolation, Fluorescence, Cell Culture