recombinant human mmp13 protein  (R&D Systems)

Journal: Biomedicines

Article Title: Fibroblast Activation Protein (FAP)-Mediated Cleavage of Type III Collagen Reveals Serum Biomarker Potential in Non-Small Cell Lung Cancer and Spondyloarthritis

doi: 10.3390/biomedicines12030545

Figure Lengend Snippet: Assay specificity: ( a ) Standard curves of signal inhibition relative to background (B/B0) with increasing peptide concentrations for 30aa selection peptide (std) (AGPAGAPGPAGSRGAPGPQGPRGDKGETGE), 10aa selection peptide (AGPAGAPGPA), deselection peptide (De) 1, 2, and 3 (AGPAGAAGQP, PGPAGAPGPA, and AGPQGAPGPA, respectively), elongated peptide (PAGPAGAPGPA), and truncated peptide (_GPAGAPGPA). Dotted lines indicate measurement range. ( b , c ) Biomarker measurements of C3F ( b ) and C3M ( c ) in solution of recombinant type III collagen (COL3) after incubation with/without matrix metalloproteinases 9 (MMP9) or fibroblast activation protein (FAP), with buffer as control. ( d , e ) Biomarker measurements of C3F ( d ) and C3M ( e ) in solution of recombinant type III collagen pre-cleaved with MMP13 (pre-cleaved COL3) followed by incubation with/without MMP9 or FAP, with buffer as control. Dotted lines indicate lower limit measurement range (LLMR) for C3F ( b , d ) and upper limit measurement range (ULMR) for C3M ( c , e ).

Article Snippet: In addition, we incubated recombinant type III collagen (Sigma-Aldrich, St. Louis, MO, USA, cat. #CC054) with MMP9 (Bio-Techne, Minneapolis, MN, USA, cat. #911-MP) or MMP13 (bio-techne cat. #511-MM) for 24 h and stopped the reaction with EDTA.

Techniques: Inhibition, Selection, Biomarker Assay, Recombinant, Incubation, Activation Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A. Paraffin sections prepared from the colons of Control, CR or CR+DBZ group of Dclk1 ΔIEC mice were subjected to IMC. MMP13 (green) is overlayed with DCLK1 (red) and DNA (blue). Boxed areas in CR group indicates significant co-localization of MMP13 with DCLK1. Scale bars as indicated (100 μ m); 8-10 mice /group. B. Box plots of DCLK1 and MMP13 counts based on IMC data set in the Control, CR, CR+DBZ groups. C. DCLK1 and MMP13 staining from IMC in control and Dclk1-S OE group after DSS-induced colitis. Lane 1 DCLK1(red) staining is overlayed with DNA (blue), Lane 2 MMP13 (red) staining is overlayed with DNA (blue), Lane 3 DCLK1(red)/MMP13(green)/DNA(Blue) colocalization in the Dclk1-S OE mice. P values as indicated. D. MMP13 promoter-reporter activity (*, ** p <0.05; n = 3 independent experiments). Ei. Western blot data of HCT116 colon cancer cells treated with PMA and PMA+DBZ. Eii. In silico molecular docking studies to predict MMP13 and DCLK1 binding. CR: Citrobacter rodentium , CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ), PMA: Phorbol 12-Myristate 13-Acetate.

Article Snippet: To examine DCLK1 binding/kinase activity, we used recombinant human DCLK1 protein (full length, rhDCLK1 (D14-10H, Sino Biologicals) and recombinant human MMP13 protein, rhMMP13 Pro form (511-MM-010, R&D Systems).

Techniques: Control, Staining, Activity Assay, Western Blot, In Silico, Binding Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: RKO cells were treated with PMA or PMA plus selective and potent MMP13 inhibitor WAY 170523 at varying doses as indicated for 30 min followed by measurement of enzymatic activity. A. Reference curve showing relative fluorescence units (RFUs). B, C. Dose-dependent decrease in MMP13 enzymatic activity (n = 3 independent experiments; * p <0.05). D. Western blots showing relative protein abundance in three colon cancer cell lines. Boxed area represents levels of the indicated proteins in RKO cells. E. Subcellular compartmentalization of proteins from RKO cells. 1: Cytosolic Fraction, 2: Nuclear Fraction, 3: Membrane Fraction, 4: Cytoskeletal fraction (n = 3 independent experiments). F. Promoters for DCLK1-L and DCLK1-S isoforms were cloned and transfected in HEK293 cells and promoter-reporter activity assays were performed using Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI). Luminescence was measured using a BioTek Synergy Neo luminometer. P values as indicated; n = 3 independent experiments.

Article Snippet: To examine DCLK1 binding/kinase activity, we used recombinant human DCLK1 protein (full length, rhDCLK1 (D14-10H, Sino Biologicals) and recombinant human MMP13 protein, rhMMP13 Pro form (511-MM-010, R&D Systems).

Techniques: Activity Assay, Fluorescence, Western Blot, Quantitative Proteomics, Membrane, Clone Assay, Transfection, Luciferase, Reporter Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A-D. Molecular docking study showing the interaction of proMMP13 (A) and mature MMP13 (C) with DCLK1-S. B. Ser 73 and Ser 114 highlighted in green in proMMP13, represent the phosphorylation sites for DCLK1-S. D. Arg207 and Gln211 are donor sites from MMP13 in mature protein close to Thr197 (circled). E. Both rhMMP13 and rhDCLK1 were incubated in kinase buffer in presence or absence of ATP at 30 °C for 1 hr. rhMMP13 didn’t show any autophosphorylation in the same condition. Phosphorylation was detected by pan phospho-Serine/Threonine antibody. F. rhMMP13 was incubated with rhDCLK1 in presence of ATP at 30 °C for 1 hr. Phosphorylated rhMMP13 was detected with immunoblotting using pan phospho-Serine/Threonine antibody. Representative immunoblots showing the levels of phosphorylation of rhMMP13. Right panel represents the Coomassie staining (n = 3 independent experiments). G, H. Protein band intensity ratios, pMMP13/pDCLK1 and pMMP13/Total MMP13, showing rhMMP13 phosphorylation by rhDCLK1.

Article Snippet: To examine DCLK1 binding/kinase activity, we used recombinant human DCLK1 protein (full length, rhDCLK1 (D14-10H, Sino Biologicals) and recombinant human MMP13 protein, rhMMP13 Pro form (511-MM-010, R&D Systems).

Techniques: Phospho-proteomics, Incubation, Western Blot, Staining

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A. Tissue sections prepared from the colons of indicated groups of mice were subjected to immunostaining with antibodies against MMP13 (red), α -SMA (green), Collagen (green), E-cadherin (green) and Vimentin (green). Samples were analyzed using the Hyperion Imaging System (Standard BioTools). DAPI (blue) was used to label DNA. Scale bars = 100 μ m (n = 8-10mice/group). B. Box plots of MMP13 and Collagen counts based on IMC data set in the Control, CR and CR+DBZ groups. C. An overlay of Collagen (Red) and MMP13 (green) in tissue sections of the indicated groups. The boxed area represents a magnified image of collagen accumulation in the CR+DBZ group. Scale bars = 100 μ m (n = 8-10mice/group). D. t-SNE plots showing MMP13 intensity across different groups. E. Masson’s Trichrome staining of tissue sections prepared from the colons of Control, CR, and CR+DBZ mice. Please note increases in collagenous fibrous tissue (stained blue) in both CR and CR+DBZ groups. Scale bars = 200 μ m; n = 8-10mice/group. CR: Citrobacter rodentium , CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ).

Article Snippet: To examine DCLK1 binding/kinase activity, we used recombinant human DCLK1 protein (full length, rhDCLK1 (D14-10H, Sino Biologicals) and recombinant human MMP13 protein, rhMMP13 Pro form (511-MM-010, R&D Systems).

Techniques: Immunostaining, Imaging, Control, Staining

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Primary antibodies for western bolt

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Primer sequence used in the RT-qPCR experiment

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Sequencing

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: MA inhibited IL-1β-stimulated ECM degeneration in NPCs. ( A - D ) The expressions of anabolic-related proteins (Aggrecan, Collagen II, and Collagen I) and catabolic-related proteins (Mmp2, Mmp3, Mmp9, and Mmp13) in NPCs were analyzed with western blotting. ( E - G ) the expressions of Collagen II and Mmp13 were detected by immunofluorescence, scalebar = 200 μm. ( H ) The mRNA expression of Collagen II and Mmp13 were analyzed by RT-qPCR. Data were shown as means ± SD, n = 3. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Western Blot, Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Inhibition of Ppar-γ weakened the anti-inflammatory, anabolism enhancing, and catabolism suppressing effects of MA on IL-1β- stimulated NPCs. ( A - B ) The proteins expressions of Ppar-γ were analyzed with western blotting. ( C - D ) The expressions of anabolic proteins (Collagen II and Collagen I) and catabolic protein (Mmp13) in the Ppar-γ-inhibition NPCs along with or without the administration of 5 ng/ml of IL-1β and 80 µM of MA. ( E ) The mRNA expression of Collagen II, Mmp13, Cox-2, and Inos were detected by RT-qPCR analysis. Data were shown as means ± SD, n = 3. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Inhibition, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: MA ameliorated IVDD-related change in rat IVDD models. ( A ) The representative X-ray images and MRI images ( B ) the measurement of DHI and the evaluation of Pfirrmann score among the SHAM, IVDD, and IVDD + MA groups. ( C ) H&E staining, ( D ) Safranin O/Fast Green staining. ( E ) IHC images of Aggrecan, Mmp13, Collagen II, Ppar-γ, and ( F ) the quantification of positive NPCs. Data were shown as means ± SD, n = 6. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Staining

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Primary antibodies for western bolt

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Primer sequence used in the RT-qPCR experiment

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Sequencing

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: MA inhibited IL-1β-stimulated ECM degeneration in NPCs. ( A - D ) The expressions of anabolic-related proteins (Aggrecan, Collagen II, and Collagen I) and catabolic-related proteins (Mmp2, Mmp3, Mmp9, and Mmp13) in NPCs were analyzed with western blotting. ( E - G ) the expressions of Collagen II and Mmp13 were detected by immunofluorescence, scalebar = 200 μm. ( H ) The mRNA expression of Collagen II and Mmp13 were analyzed by RT-qPCR. Data were shown as means ± SD, n = 3. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Western Blot, Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: MA suppressed MAPK and NF-κB pathways in IL-1β-stimulated NPCs. The proteins expressions of NF-κB (P65) ( A - B ) and MAPK (P38, Jnk, and Erk) ( E - F ) were analyzed with western blotting. ( C ) Immunofluorescence staining and ( D ) quantitative analysis of the nuclear translocation of P-P65 in NPCs. Data were shown as means ± SD, n = 3. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Western Blot, Immunofluorescence, Staining, Translocation Assay

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Inhibition of Ppar-γ weakened the anti-inflammatory, anabolism enhancing, and catabolism suppressing effects of MA on IL-1β- stimulated NPCs. ( A - B ) The proteins expressions of Ppar-γ were analyzed with western blotting. ( C - D ) The expressions of anabolic proteins (Collagen II and Collagen I) and catabolic protein (Mmp13) in the Ppar-γ-inhibition NPCs along with or without the administration of 5 ng/ml of IL-1β and 80 µM of MA. ( E ) The mRNA expression of Collagen II, Mmp13, Cox-2, and Inos were detected by RT-qPCR analysis. Data were shown as means ± SD, n = 3. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Inhibition, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: Inhibition of Ppar-γ weakened the inhibition effect of MA on NF-κB signaling pathway in IL-1β-stimulated NPCs. ( A - D ) Western blot analysis showed the inhibition of Ppar-γ activated the NF-κB (P65) and MAPK (P38, Jnk, and Erk) signaling pathway. Data were shown as means ± SD, n = 3. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Inhibition, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Mulberroside A mitigates intervertebral disc degeneration by inhibiting MAPK and modulating Ppar-γ/NF-κB pathways

doi: 10.1186/s12950-024-00398-7

Figure Lengend Snippet: MA ameliorated IVDD-related change in rat IVDD models. ( A ) The representative X-ray images and MRI images ( B ) the measurement of DHI and the evaluation of Pfirrmann score among the SHAM, IVDD, and IVDD + MA groups. ( C ) H&E staining, ( D ) Safranin O/Fast Green staining. ( E ) IHC images of Aggrecan, Mmp13, Collagen II, Ppar-γ, and ( F ) the quantification of positive NPCs. Data were shown as means ± SD, n = 6. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significant difference

Article Snippet: Following a rinse with Tris Buffered Saline with Tween (TBST), the cells were fixed with a fixation reagent (Servicebio, China) for 0.5 h and then permeabilized with 0.2% Triton X-100 (BioFroxx, Germany) for 0.1 h. After being blocked for 1 h, the NPCs were incubated with the primary antibodies (Mmp13, Collagen II, and p-P65, 1:200) at 4 °C for more than 16 h. Subsequently, corresponding fluorescent secondary antibodies (Boster, China) were added to the cells and incubated for 1 h away from light.

Techniques: Staining

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Proposed action mechanism of ERMs@siM13 in the early-stage posttraumatic osteoarthritis (PTOA) mouse model. (A) Schematic illustration of preparation procedure of ERMs@siM13 and its matrix metalloproteinase 13 (MMP13)-mediated activation. (B) After intraarticular injection into the joints with early-stage PTOA, ERMs@siM13 can be activated by MMP13 overexpressed surrounding diseased chondrocytes, detaching the Cy5-bearing PEG protective shell. Accordingly, cRGD is exposed to promote the siRNA silencing MMP13 (siM13)-loaded micelle uptake by the diseased cells for on-demand MMP13 downregulation, and Cy5 fluorescence is restored to report the diseased state of cartilage tissues.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Activation Assay, Injection, Fluorescence

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Preparation and characterization of ERMs@siM13. (A) High-performance liquid chromatography (HPLC) spectra of Cy5-C(PEG 2000 )-GPLGVRGK-NH 2 (EP) and Cy5-C(PEG 2000 )-GplgvrGK-NH 2 (nEP) before and after incubation with matrix metalloproteinase 13 (MMP13). (B) Representative flow cytometry histogram showing the uptake of coumarin 6 (C6)-labeled RMs with various cRGD modification densities by diseased chondrocytes and (C) the corresponding mean fluorescence intensity (MFI) of C6. (D) Representative flow cytometry histogram showing the internalization of nERMs@C6 with various ratios of cRGD: PEG by diseased chondrocytes and (E) the corresponding C6 MFI. (F) Representative fluorescence spectra and fluorescence images of nERMs containing various molar ratios of BHQ3-to-Cy5. (G) Representative transmission electron microscopy (TEM) images of ERMs@siM13. Scale bar: 50 nm. (H) Representative fluorescence spectra and fluorescence images of ERMs and nERMs before and after incubation with MMP13. (I) Serum stability of siM13 loaded by RMs, ERMs, and nERMs determined through agarose gel electrophoresis. Free siM13 served as the controls. “E” represents MMP13. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: High Performance Liquid Chromatography, Incubation, Flow Cytometry, Labeling, Modification, Fluorescence, Transmission Assay, Electron Microscopy, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: ERMs@siM13 delivery at cellular levels. (A) Schematic illustration of MMP13-triggered diseased chondrocyte-specific delivery and fluorescence imaging of ERMs@siM13. (B) Fold change of MMP13 expression in culture medium was determined by enzyme-linked immunosorbent assay (ELISA) after chondrocytes were induced by IL-1β for different intervals. (C) Representative fluorescence images and the corresponding fluorescence intensity of normal or diseased chondrocytes after incubation with ERMs or nERMs for 2 h. (D) Representative flow cytometry histogram showing the uptake of coumarin 6 (C6)-labeled micelles by the diseased chondrocytes after 2 h of incubation and the corresponding mean fluorescence intensity (MFI) of C6. (E) Representative confocal images of IL-1β-induced diseased chondrocytes after 2 h of incubation with C6-labeled various micelles. Scale bar: 50 μm. (F) Intracellular delivery of ERMs@FAM-siRNA in IL-1β-induced diseased chondrocytes. Late endosomes/lysosomes are labeled by LysoTracker Red. Scale bar: 25 μm. (G) Pearson's correlation coefficients calculated from Panel F. Data are presented as mean ± SD (n = 3). ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Fluorescence, Imaging, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Labeling, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Diagnostic and therapeutic effects of ERMs@siM13 on diseased chondrocytes. (A) The diseased chondrocytes were treated with the indicated formulations, followed by 2 h of incubation with ERMs to diagnose the chondrocyte status. Normal chondrocytes served as a control. (B) Mean fluorescence intensity (MFI) of Cy5 calculated from Panel (A). (C) Mmp1 3 mRNA levels of diseased chondrocytes relative to that of the PBS control after treated with RMs@siM13, nERMs@siM13, and ERMs@siM13. (D) Representative Western blot images showing MMP13, Col 2, and GAPDH protein levels after treatment mentioned above and the corresponding mean protein expression of MMP13 and Col 2. (E–F) Immunofluorescence staining and semiquantitative analysis of (E) MM13 and (F) Col 2 for diseased chondrocytes after treatment mentioned above. (G) Proliferation assay of diseased chondrocytes that were treated as mentioned above, as determined via EdU staining. EdU + cell ratios relative to the PBS control were calculated. Scale bar: 100 μm. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Diagnostic Assay, Incubation, Control, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Proliferation Assay, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: In vivo delivery and diagnostic effects of ERMs@siM13 in the murine posttraumatic osteoarthritis (PTOA) model. (A) Representative fluorescence images of the PTOA mouse joints at various time points after intraarticular injection with ERMs@DiR and nERMs@DiR. (B) The fluorescence signal from Panel (A) was qualified. (C) Representative confocal images showing the ERMs@C6 and nERMs@C6 penetration into healthy and PTOA cartilage. (D) Mean fluorescence intensity (MFI) of C6 calculated from Panel (C). (E) The PTOA mice were treated with ERMs@siM13 and nERMs@siM13 every 5 days for 9 cycles. ERMs were intraarticularly injected at preset time points to monitor the PTOA progression. (F) Cy5 MFI calculated from Panel (E). (G) The PTOA mice were treated as described above. The joints were collected at preset time points and processed for immunofluorescence staining of MMP13. (H) MMP13 MFI calculated from Panel (G). Scale bar: 100 μm. Data are presented as mean ± SD. * P < 0.05, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: In Vivo, Diagnostic Assay, Fluorescence, Injection, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Immunohistochemical (IHC) staining of sham or posttraumatic osteoarthritis (PTOA) mouse knee joints after 4 and 8 weeks of treatment. (A) Representative IHC staining images of MMP13 in the mouse cartilages after treatment with the indicated formulations. The sham group served as a normal control. (B) The MMP13-positive (MMP13 + ) area (%) calculated from Panel (A). (C) Representative IHC staining images of Col 2 in the mouse cartilages posttreatment with the indicated formulations. (D) The Col 2-positive (Col 2 + ) area calculated from Panel (C). (E) Representative IHC staining images of aggrecan (ACAN) in the mouse cartilages posttreatment with the indicated formulations. (F) The ACAN-positive (ACAN + ) area calculated from Panel (E). Scale bar: 100 μm. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Control, Two Tailed Test

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 1. FAPs from MMP13 KO mice have increased spontaneous adi- pogenesis and decreased sponta- neous fibrogenesis compared to FAPs from wildtype mice cultured in a standard medium. A) A typical image of immunostaining for peril- ipin A and αSMA for FAPs from WT and MMP13 KO mice after 2 weeks of culture in standard medium Bottom: negative control of perili- pin A immunofluorescence stain- ing (without primary antibody), negative control of αSMA immu- nofluorescence staining (with- out primary αSMA antibody). B) FAPs from MMP13 KO mice had a significantly higher percentage of perilipin A positive cells when compared to FAPs from WT mice. C) FAPs from MMP13 KO mice had significantly lower αSMA positive cells when compared to FAPs from WT mice. D) Real time PCR showed that FAPs from MMP13 KO mice had significantly higher expression of Adiponectin, but a lower expres- sion of Collagen I compared to FAPs from wildtype mice (* p<0.05).

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Cell Culture, Immunostaining, Negative Control, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 2. Wildtype FAPs treated with MMP13 inhibitor had sig- nificantly increased adipogen- esis. A) A Typical image of immu- nostaining for FAPs treated with 10μM MMP13 inhibitor and 0.1% DMSO in standard medium for 2 weeks. B) Wildtype FAPs treated with MMP13 inhibitor had a sig- nificantly higher percentage of perilipin A(+) cells compared to those treated with DMSO. C) FAPs treated with MMP13 inhibitor had a significantly reduced number of αSMA(+) cells compared to those treated with DMSO. D) Real time PCR showed that FAPs treated with the MMP13 inhibitor had a sig- nificantly increased expression of Adiponectin, PPARγ, C/EBPA and deceased expression of αSMA and Collagen 1a (* p<0.05).

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 3. MMP13 treatment inhibits FAP adipogenesis and promotes FAP fibrogenesis. A) A typical im- age of immunostaining for FAPs treated with 100 ng/ml MMP13 and 0.1% DMSO in standard me- dium for 2 weeks. B) FAPs treated with MMP13 have a significantly reduced the number of perili- pin (+) cells compared to DMSO. C) FAPs treated with MMP13 have a significantly increased the num- ber of αSMA (+) cells compared to DMSO. D) Real time PCR results of FAPs treated with MMP13 have a significantly higher expression of αSMA and collagen I and decreased expression of Adiponectin and PPARγ (* p<0.05).

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Immunostaining, Real-time Polymerase Chain Reaction, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 4. A) The typical images of FAPs from wildtype and MMP13 KO mice treated with TGFβ-1, TGFβ in- hibitor, or 0.1% DMSO in a standard medium for 2 weeks. B) TGFβ-1 significantly decreased the number of perilipin (+) cells and the TGFβ inhibitor significantly increased the number of perilipin (+) cells in FAP WT mice, but not in FAPs from MMP13 KO mice. C) TGFβ-1 significantly increased the number of αSMA (+) cells and the TGFβ inhibitor significantly decreased the number of αSMA (+) cells in FAPs from WT mice, but not in FAPs from MMP13 KO mice. D) & E) Real time PCR results showed that TGFβ-1 significantly decreased the expression of adipogenesis-related genes and increased fibrogenesis-related gene expression of FAPs in WT mice, while the TGFβ inhibitor had an opposite effect. However, the effect of the TGFβ-1 and TGFβ inhibitors had no effect on their expression in FAPs from MMP13 KO mice ( * p<0.05). F) Exogenous MMP13 added to the TGFβ inhibitor treatment group in both WT mice and MMP13 KO mice. Exogenous MMP13 added to the MMP-13 KO mice as a control. G) Quantification of the percentage of the number of Perilipin A(+) cells and αSMA (+) cells out of total number of cells.

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 5. A) Typical images of FAPs from WT and MMP13 KO mice treated with BMP-7, BMP inhibitor, or 0.1% DMSO in standard medium for 2 weeks. B) BMP-7 significantly increased the number of perilipin (+) cells and the BMP inhibitor significantly decreased the number of perilipin (+) cells in FAPs in WT mice, but not in FAPs from MMP13 KO mice. C) BMP-7 significantly decreased αSMA (+) cells and the BMP inhibitor sig- nificantly increased the number of αSMA (+) cells in FAPs in WT mice, but not in FAPs from MMP13 KO mice. D) & E) Real time PCR results showed that BMP-7 significantly increased the expression of adipogenesis- related genes and decreased fibrogenesis-related gene expression of FAPs in WT mice, while BMP inhibitor had an opposite effect. Neither BMP-7 nor BMP inhibitor showed an effect on adipogenesis-related and fibrogenesis-related gene expression in FAPs from MMP13 KO mice ( * p<0.05). F) Exogenous MMP13 added to the BMP inhibitor treatment group in both WT mice and MMP13 KO mice. Exogenous MMP13 was added to the MMP13 KO mice as a control. G) Quantification of Perilipin A and αSMA.

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 6. A) Typical images of FAPs from WT mice treated with the MMP13 inhibitor (or DMSO) in combina- tion of TGFβ-1 and TGFβ inhibitor in standard medium for 2 weeks. B) TGFβ-1 significantly decreased the number of perilipin A(+) cells and TGFβ inhibitor significantly increased the number of perilipin A (+) cells in FAPs without the MMP13 inhibitor. However, MMP13 inhibitor blocked the effect of TGFβ-1 and TGFβ inhibitors. C) TGFβ-1 significantly increased the numbers of αSMA (+) cells and the TGFβ inhibitor signifi- cantly decreased the number of αSMA (+) cells in FAPs without the MMP13 inhibitor. However, the MMP13 inhibitor blocked the effects of the TGFβ-1 and TGFβ inhibitor. D) & E) Real time PCR results showed that TGFβ-1 significantly decreased adipogenesis-related gene expression and increased fibrogenesis-related gene expression of FAPs without the MMP13 inhibitor, but not in combination with the MMP13 inhibitor (* p<0.05).

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

doi: 10.33594/000000596

Figure Lengend Snippet: Fig. 7. A) Typical images of FAPs from WT mice treated with an MMP13 inhibitor (or DMSO) in combination with BMP-7 and the BMP inhibitor in standard medium for 2 weeks. B) BMP-7 significantly increased the number of perilipin (+) cells and BMP inhibitor significantly decreased the number of perilipin (+) cells in FAPs without the MMP13 inhibitor, but not in combination with the MMP13 inhibitor. C) BMP-7 significantly decreased the number of αSMA (+) cells and the BMP inhibitor significantly increased the number of αSMA (+) cells in FAPs from WT mice, but not in FAPs from MMP13 KO mice. D) & E) Real time PCR results showed that BMP significantly increased the expression of adipogenesis-related genes and decreased the expression of fibrogenesis-related in FAPs from WT mice, while the BMP inhibitor had an opposite effect. However, the effect of BMP-7 and BMP inhibitor was abolished in FAPs from MMP13 KO mice (* p<0.05).

Article Snippet: MMP13 treatment Recombinant Human MMP13 (R&D systems, 511-MM-010, Minneapolis, MN, USA) were first activated with 1 μM APMA p-Aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563, MO, USA) at 37°C for 2 hours.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer letters

Article Title: Elevated MMP10/13 mediated barrier disruption and NF-κB activation aggravate colitis and colon tumorigenesis in both individual or full miR-148/152 family knockout mice.

doi: 10.1016/j.canlet.2021.12.033

Figure Lengend Snippet: Fig. 3. Loss of miR-148/152 family members increases MMP10 and MMP13. A. Venn diagram representing overlap of differentially expressed genes between the GEO dataset and TCGA database. B. Venn diagrams representing overlapping differentially expressed genes between miR-148/152 family target genes and upre gulated genes screened from the GEO dataset and TCGA database. C-D. Expression of MMP10 and MMP13 was detected by real-time PCR and western blotting after transfection with miR-148/152 family member inhibitors or mimics. E-F. Luciferase activity in Caco-2 cells. G. Western blotting detecting protein expression of target genes in mice. H–I. Representative IHC images of MMP10 and MMP13 immunostaining in colon sections, scale bar: 50 μm. Data in this figure are presented as mean ± SD. C, E-F analyzed via t-test.

Article Snippet: Recombinant MMP10 or MMP13 (R&D systems, USA) were diluted to 50 mg/ml in TCNB buffer (FEIYUBIO, China) with 1 mM 4-aminophenylmercuric acetate (APMA), then activated via incubating for 2 h at 37 ◦C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Activity Assay, Immunostaining

Journal: Cancer letters

Article Title: Elevated MMP10/13 mediated barrier disruption and NF-κB activation aggravate colitis and colon tumorigenesis in both individual or full miR-148/152 family knockout mice.

doi: 10.1016/j.canlet.2021.12.033

Figure Lengend Snippet: Fig. 5. Loss of miR-148/152 family members causes hyperactivation of the NF-κB signaling pathway. A. Western blotting showing MMP10 and MMP13 both shear proTNF-α to generate bioactive TNF-α fragments. B. Activation of NF-κB pathway members was examined in mice with induced colitis and CAC via western blotting. C. IHC detecting p-p65 nuclear translocation to show NF-κB signaling pathway activity, scale bar: 50 μm. D. Venn diagram for target genes in the NF-κB signaling pathway. E-F. Expression of IKKα and IKKβ detected by real-time PCR and western blotting after transfection with miR-148/152 family member inhibitors or mimics. G. Western blotting detecting protein expression of IKKα and IKKβ in mice. H. Luciferase activity in Caco-2 cells. Data in this figure are presented as mean ± SD. E, H analyzed via t-test.

Article Snippet: Recombinant MMP10 or MMP13 (R&D systems, USA) were diluted to 50 mg/ml in TCNB buffer (FEIYUBIO, China) with 1 mM 4-aminophenylmercuric acetate (APMA), then activated via incubating for 2 h at 37 ◦C.

Techniques: Western Blot, Shear, Activation Assay, Translocation Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase

Journal: Cancer letters

Article Title: Elevated MMP10/13 mediated barrier disruption and NF-κB activation aggravate colitis and colon tumorigenesis in both individual or full miR-148/152 family knockout mice.

doi: 10.1016/j.canlet.2021.12.033

Figure Lengend Snippet: Fig. 9. Deleting the miR-148/152 family promotes colitis and colon tumorigenesis via barrier disruption and NF-κB activation. In homeostasis, miR-148/ 152 family members impede MMP10, MMP13, IKKα, and IKKβ, all of which can disrupt intestinal barrier and promote activation of the NF-κB signaling pathway. Deletion of miR-148/152 family members causes intestinal barrier dysfunction by enhancing MMP10 and MMP13, thereby increasing sensitivity to colitis. MMP10 and MMP13 activate proTNF-α, which is released during colitis to promote activation of the NF-κB signaling pathway. Accumulation of IKKα and IKKβ caused by loss of miR-148/152 family members results in hyperactivation of the NF-κB signaling pathway, which exacerbates colitis and CAC.

Article Snippet: Recombinant MMP10 or MMP13 (R&D systems, USA) were diluted to 50 mg/ml in TCNB buffer (FEIYUBIO, China) with 1 mM 4-aminophenylmercuric acetate (APMA), then activated via incubating for 2 h at 37 ◦C.

Techniques: Disruption, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: dsRNA Induced IFNβ-MMP13 Axis Drives Corneal Wound Healing

doi: 10.1167/iovs.63.2.14

Figure Lengend Snippet: MMP13 promotes LSCs proliferation and migration. ( A ) Pie chart showing 192 genes of group III are annotated as interferon regulated genes. ( B ) Top 10 interferon regulated genes preferentially upregulate in wounded cornea. The size of the dot represents the gene expression. ( C ) Representative immunofluorescence staining of P63, PAX6, and Ki67 expression in LSCs. Scale bar, 50 µm. ( D ) The qPCR analysis of top 10 genes expression in recombinant IFNβ treated LSCs ( n = 3). Data are represented as mean ± SE. (E) Western blot analysis of MMP13 expression in recombinant IFNβ treated LSCs at indicated time points. ( F ) FACS analysis of carboxyfluorescein succinimidyl ester (CFSE) labeled LSCs. The original fluorescence intensity at d0 represents the starting point. The control group and recombinant MMP13 treated group are analyzed at d3. Representative data from three independent experiments are shown. ( G ) Scratch wound assays of control LSCs and recombinant MMP13 treated LSCs. Representative images of LSCs at indicated time-points. Scale bar, 500 µm. ( H ) Quantifications of wound closure of LSCs are shown (Students t -test, n = 3). Data are represented as mean ± SE (* P < 0.05).

Article Snippet: The recombinant human MMP13 (rhMMP13; R&D Systems, 511-MM-010) were added to the culture medium with a concentration of 1 μg/mL.

Techniques: Migration, Gene Expression, Immunofluorescence, Staining, Expressing, Recombinant, Western Blot, Labeling, Fluorescence, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: dsRNA Induced IFNβ-MMP13 Axis Drives Corneal Wound Healing

doi: 10.1167/iovs.63.2.14

Figure Lengend Snippet: MMP13 enhances corneal wound closure. ( A ) The qPCR analysis of Mmp13 in unwounded and wounded cornea at d1 post-injury. ( B ) Representative immunofluorescence staining of MMP13 and KRT6 in unwounded and wounded cornea at d1 post-injury. Scale bar, 100 µm. ( C ) Western blot analysis of MMP13 proteins in unwounded cornea and wounded cornea treated with or without IFNβ antibody. Representative immunoblots are presented, n = 3. ( D ) Representative images of the epithelial defect in wounded corneas of control, MMP13 antibody, IFNβ antibody and IFNβ antibody combined with recombinant MMP13 treatment (for each treatment: upper panel , white light micrograph; lower panel , fluorescein staining of corneal surface). ( E ) Quantification of re-epithelialization in control, MMP13 antibody, IFNβ antibody, and IFNβ antibody combined with recombinant MMP13 treated mice corneas. Epithelial defect is presented as the percentage of the original wound size (Students t -test, n = 5). Data are represented as mean ± SE (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The recombinant human MMP13 (rhMMP13; R&D Systems, 511-MM-010) were added to the culture medium with a concentration of 1 μg/mL.

Techniques: Immunofluorescence, Staining, Western Blot, Control, Recombinant

Journal: Investigative Ophthalmology & Visual Science

Article Title: dsRNA Induced IFNβ-MMP13 Axis Drives Corneal Wound Healing

doi: 10.1167/iovs.63.2.14

Figure Lengend Snippet: Corneal wound healing process. (1) The dsRNA is released from damaged cells in wounded cornea, (2) the dsRNA triggers IFNβ expression in corneal epithelium, and (3) MMP13 exerts as a downstream effector of IFNβ and promotes corneal epithelial cells proliferation and migration.

Article Snippet: The recombinant human MMP13 (rhMMP13; R&D Systems, 511-MM-010) were added to the culture medium with a concentration of 1 μg/mL.

Techniques: Expressing, Migration