Fig 1: HGF induces upregulation of MMP1 and MMP10 expression by human corneal epithelial cells (A) Map of the matrix metalloproteinases protein array. (B, C) Human corneal epithelial cells and human corneal fibroblasts were cultured with or without HGF (50 ng/mL) for 24 h. Supernatants were harvested and expression levels of MMPs were quantified using a protein array. (B) Representative bar chart showing fold change in expression levels of MMP1 and MMP10 by human corneal epithelial cells. (C) Representative bar chart showing fold change in expression levels of MMP1 and MMP10 by human corneal fibroblasts. Fold change was calculated from expression levels of cells cultured in media alone. Mean ± SD. t-test; **p < 0.01.
Fig 2: Topical administration of HGF reduces αSMA expression and upregulates expression of MMP1 and MMP10 in injured corneas. Following scar formation (day 10 post-injury), corneas were treated with 5 μL of 0.1% HGF and 0.1% steroid (Dexamethasone), thrice a day for 10 days (days 10 to 20). 0.1% control protein served as treatment control. Corneas were harvested on day 20 post-injury. (A) Representative corneal cross-sections (left) immunostained with αSMA (green) (Scale bars, 50 μm). (B) Relative mRNA expression of MMP1 and MMP10 in lysates of harvested corneas, as quantified by real-time PCR. Data from three independent experiments are shown, and each experiment consists of 3–4 mice/group. The values shown are Mean ± SEM. t-test; *p < 0.05.
Fig 3: TNFα induces the expression of MMPs in OA chondrocytes via NF-κB activation. (A) Chondrocytes were treated with vehicle or 10 ng/ml TNFα for 24 h. MMP protein expression levels in stimulated cell lysates were semi-quantified via a human MMP antibody array (n=2). (B) MMP mRNA expression in stimulated cells was determined by reverse transcription-quantitative PCR. (C) TNFα-treated chondrocytes were assessed by immunoblotting. Phosphorylation was semi-quantified with ImageJ and calculated relative to each total protein. (D) Proteins secreted in the cell supernatant by TNFα stimulation were precipitated with trichloroacetic acid and detected by immunoblotting. Coomassie blue staining was used as a loading control. (E) 293T cells were transfected with p65 wild type or double mutants (S536S or S536E), followed by treatment with vehicle, 10 or 25 ng/ml TNFα for 24 h; promoter activity analysis was subsequently performed. n=3, one-way ANOVA with Tukey's post hoc test. (F) Protein expression levels of p-NF-κB, total NF-κB and MMP1, 3 and 13 in TNFα-stimulated cells were analyzed by immunofluorescence. Representative data are shown. Scale bar, 50 µm. *P<0.05, **P<0.01; ***P<0.001 (mean ± SD; n=6). OA, osteoarthritis; phos, phosphorylated; TNFα, tumor necrosis factor α; MMP, matrix metalloproteinase; NF-κB, nuclear factor κB.
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