Journal: bioRxiv

Article Title: Loss of mutually protective effects between osteoclasts and chondrocytes in damaged joints drives osteoclast-mediated cartilage degradation via matrix metalloproteinases

doi: 10.1101/2021.01.03.425116

Figure Lengend Snippet: (a,b) Effect of bafilomycin, E64 and GM6001 on osteoclast-mediated release of GAG from (a) acellular or (b) cellular cartilage after 13 days of differentiation. N varies for each graph, as represented by the number of data points. (c) Effect of recombinant human TIMP1 (100 nm) on GAG release from osteoclasts cultured on acellular cartilage, n=18. *, p<0.05; ***, p<0.0001.

Article Snippet: For inhibitor experiments, mature osteoclasts were treated with bafilomycin (Cell Signalling Technology, Hitchin, UK), E64 (Cambridge Bioscience, Cambridge, UK), GM6001 (Selleckchem, Munich, Germany) or recombinant human TIMP1 (R&D Systems).

Techniques: Recombinant, Cell Culture

Journal: Physiological Reports

Article Title: Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis

doi: 10.1002/phy2.125

Figure Lengend Snippet: Effects of BAPTA/AM and nocodazole on TIMP-1 secretion. Media were collected from LX-2 cells 30 min after no treatment or treatment with (A) BAPTA/AM (50 μmol/L) and/or (B) nocodazole (20 μmol/L). TIMP-1 levels were determined by ELISA and for (B) normalized to baseline TIMP-1 secretion. BAPTA/AM and nocodazole decreased TIMP-1 secretion to ∼40–75% of baseline (* P < 0.05; ** P < 0.01), and the effects of BAPTA/AM and nocodazole were not additive. Furthermore, nocodazole did not reduce TIMP-1 secretion beyond that of BAPTA/AM alone ( P = 0.12). ( n ≥ 3 for each condition).

Article Snippet: For total internal reflection fluorescence (TIRF) microscopy experiments, a commercially available plasmid encoding Turbo-green fluorescence protein (GFP) fluorescent probe attached to the C-terminus of human TIMP-1 protein under the control of a CMV promoter (Origene Technologies, Rockville, MD) was used.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Physiological Reports

Article Title: Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis

doi: 10.1002/phy2.125

Figure Lengend Snippet: Rapid decreases in TIMP-1 release are not mediated by changes in TIMP-1 transcription. LX-2 cells were either left untreated or treated with the calcium chelator BAPTA/AM (50 μmol/L) or the Ca 2+ i agonist hormone vasopressin (2 μmol/L). Changes in TIMP-1 mRNA were determined by real-time RT-PCR. No differences in TIMP-1 mRNA levels were noted at the 30 min time point. Interestingly, both VP and BAPTA/AM increased TIMP-1 mRNA levels at 12 h ( n = 5 for each condition; * P < 0.05).

Article Snippet: For total internal reflection fluorescence (TIRF) microscopy experiments, a commercially available plasmid encoding Turbo-green fluorescence protein (GFP) fluorescent probe attached to the C-terminus of human TIMP-1 protein under the control of a CMV promoter (Origene Technologies, Rockville, MD) was used.

Techniques: Quantitative RT-PCR

Journal: Physiological Reports

Article Title: Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis

doi: 10.1002/phy2.125

Figure Lengend Snippet: LX-2 cells transfected with TIMP-1-DsRed exhibit temporally related Ca 2+ i signals and loss of DsRed fluorescence. (A) Representative images. Unlike the distribution of DsRed seen in Figure , TIMP-1-DsRed trafficked to discrete regions within LX-2 cells in a vesicular pattern. VP induced intracellular Ca 2+ i signals similar to those seen in Figure ; however, in addition, VP induced a decrease in TIMP-1-DsRed fluorescence. 400× magnification. (B) Graphical representation of changes in Fluo-4/AM calcium indicator and DsRed fluorescence. Changes in fluorescence were determined as described in Figure . VP again induced a sustained Ca 2+ i increase, which was followed by a marked decrease in DsRed fluorescence over the subsequent 20–60 sec. (C) Representative images. Serial TIRF microscopy images of transiently transfected LX-2 cells with TIMP-1-GFP also suggest that intracellular distribution of TIMP-1-GFP proteins follows a vesicular pattern observed at the subplasmalemmar levels. Pseudocolored frames corresponding to various time points post VP stimulation ( t = 1 min, green; t = 6 min, red; t = 15 min, cyan; t = 26 min, magenta) were combined to produce the composite image labeled as “ merged frames ”. The latter image shows areas where moving (arrows) and stable (arrowheads) TIMP-1-GFP vesicles were observed. 400× magnification.

Article Snippet: For total internal reflection fluorescence (TIRF) microscopy experiments, a commercially available plasmid encoding Turbo-green fluorescence protein (GFP) fluorescent probe attached to the C-terminus of human TIMP-1 protein under the control of a CMV promoter (Origene Technologies, Rockville, MD) was used.

Techniques: Transfection, Fluorescence, Microscopy, Labeling

Journal: Physiological Reports

Article Title: Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis

doi: 10.1002/phy2.125

Figure Lengend Snippet: VP-sensitive decreases in TIMP-1-DsRed fluorescence were inhibited by calcium chelation. Aggregate changes in DsRed fluorescence ( n = 5 per condition) were determined in LX-2 cells transfected with DsRed (control) or TIMP-1-DsRed ± BAPTA/AM (50 μmol/L). The VP-sensitive decrease in DsRed fluorescence in LX-2 cells expressing TIMP-1-DsRed (* P < 0.001) was inhibited by pretreatment with BAPTA/AM.

Article Snippet: For total internal reflection fluorescence (TIRF) microscopy experiments, a commercially available plasmid encoding Turbo-green fluorescence protein (GFP) fluorescent probe attached to the C-terminus of human TIMP-1 protein under the control of a CMV promoter (Origene Technologies, Rockville, MD) was used.

Techniques: Fluorescence, Transfection, Expressing

Journal: Physiological Reports

Article Title: Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis

doi: 10.1002/phy2.125

Figure Lengend Snippet: TIMP-1 colocalizes with microtubules but not microfilaments in LX-2 cells. (A) Confocal immunofluorescence comparing distribution of endogenous TIMP-1 and α -tubulin. Localized expression of TIMP-1 and α -tubulin were determined in untransfected LX-2 cells by confocal immunofluorescence. (a) TIMP-1 fluorescence is pseudocolored red, α -tubulin fluorescence is pseudocolored green, and nuclear staining (TO-PRO) is pseudocolored blue. Focused images at the plasma membrane, either closer to the nucleus (b–d insets) or in cell extensions (e–g insets) demonstrate that endogenous TIMP-1 is concentrated in a vesicular pattern colocalizing with α -tubulin. (a) 630× magnification, (b–g) 3× zoom-in from the (a) picture. (B) Confocal immunofluorescence comparing distribution of endogenous TIMP-1 and F-actin. Localized expression of TIMP-1 and actin microfilaments were determined in untransfected LX-2 cells by confocal immunofluorescence. (a) TIMP-1 fluorescence staining is pseudocolored red, and tetramethylrhodamine-phalloidin fluorescence staining is pseudocolored green. Unlike in the left image, TIMP-1 does not appear to colocalize with filamentous actin in the perinuclear cytoplasm (b–d insets) or in cell extensions (e–g insets). (a) 630× magnification, (b–g) 3× zoom-in from the (a) picture. (C) Confocal immunofluorescence comparing distribution of endogenous TIMP-1 versus exogenous TIMP-1-GFP. LX-2 cells were transfected with a TIMP-1-GFP (green) expression vector, immunolabeled with anti-TIMP-1 (red), and stained with DAPI nuclear dye (blue). All TIMP-1-GFP proteins (b) are also labeled with anti-TIMP-1, and the majority of the vesicles observed are at or near the plasma membrane or perinuclear cytoplasm (a,b,d). Interestingly, native TIMP-1 is also noted in a vesicular pattern in an intermediate region (a,c). (a) 400× magnification, (b,c, and d) 3× zoom-in from the (a) picture. (D) Confocal immunofluorescence comparing distribution of TIMP-1-GFP and F-actin. LX-2 cells were transfected with a TIMP-1-GFP (green) expression vector and stained with tetramethylrhodamine-phalloidin (red) (a). TIMP-1-GFP-containing vesicles in the peri-nuclear cytoplasm do not colocalize with phalloidin-stained F-actin (a); however, there is near or colocalization in the region of the plasma membrane (c–d). (a) 400× magnification, (b,c, and d) 3× zoom-in from the (a) picture. (E) Confocal immunofluorescence comparing distribution of TIMP-1-GFP and α -tubulin. LX-2 cells were transfected with a TIMP-1-GFP (green) expression vector and immunolabeled with anti- α -tubulin (red) (a). No colocalization between TIMP-1-GFP and α -tubulin was observed (b–d). (a) 400× magnification, (b,c, and d) 3×zoom-in from the (a) picture. (F) Immunoblot to determine specificity of TIMP-1 antibody. The TIMP-1 antibody used for the immunofluorescence figures above was used to determine the expression of TIMP-1 in LX-2 cells transfected with TIMP-1-DsRed. The TIMP-1 antibody recognizes a 25-kDa band, representing native (or wild type) TIMP-1 (white arrowhead), and a 55–65 kDa band, representing expressed TIMP-1-DsRed fusion protein (black arrowhead). The relative intensities of these bands suggest that the majority of TIMP-1 expression in transfected LX-2 cells is exogenous.

Article Snippet: For total internal reflection fluorescence (TIRF) microscopy experiments, a commercially available plasmid encoding Turbo-green fluorescence protein (GFP) fluorescent probe attached to the C-terminus of human TIMP-1 protein under the control of a CMV promoter (Origene Technologies, Rockville, MD) was used.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Transfection, Plasmid Preparation, Immunolabeling, Labeling, Western Blot

Journal: Physiological Reports

Article Title: Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis

doi: 10.1002/phy2.125

Figure Lengend Snippet: Effects of microtubules, microfilaments, and atypical myosins inhibition on TIMP-1 exocytosis. Changes in VP-sensitive decreases in TIMP-1-DsRed fluorescence were determined in LX-2 cells pretreated with either nocodazole (20 μmol/L) for 30–60 min, cytochalasin D (2 μmol/L) for 1–2 h or varying concentrations of blebbistatin (5–100 μmol/L) for 30 min ( n = 4–5 for all experiments). (A) Effect of microtubules inhibitor nocodazole. Nocodazole completely inhibited VP-sensitive TIMP-1-DsRed exocytosis at 30–60 min ( n = 5 for all groups; P < 0.01). (B) Effect of microfilaments inhibitor cytochalasin D. Cytochalasin D partially inhibited VP-sensitive TIMP-1-DsRed exocytosis at 1 h (* P < 0.01 vs. control; % P = 0.521 vs. control) and completely inhibited VP-sensitive TIMP-1-DsRed exocytosis at 2 h. (C) Effect of atypical myosins inhibitor blebbistatin. Blebbistatin had no effect on VP-sensitive TIMP-1-DsRed exocytosis at 5 μmol/L but blocked TIMP-1-DsRed exocytosis at 50 μmol/L and 100 μmol/L.

Article Snippet: For total internal reflection fluorescence (TIRF) microscopy experiments, a commercially available plasmid encoding Turbo-green fluorescence protein (GFP) fluorescent probe attached to the C-terminus of human TIMP-1 protein under the control of a CMV promoter (Origene Technologies, Rockville, MD) was used.

Techniques: Inhibition, Fluorescence

Journal: Molecular Medicine Reports

Article Title: Gaseous signalling molecule SO 2 via Hippo-MST pathway to improve myocardial fibrosis of diabetic rats

doi: 10.3892/mmr.2017.7714

Figure Lengend Snippet: SO 2 improves myocardial fibrosis in diabetic rats. (A) Morphological changes in myocardium assessed by Masson staining. Images were acquired at ×400 magnification. Expression levels of (B) MMP9, (C) MMP24 and (D) TIMP1 in each group. Date are expressed as mean ± standard deviation (n=3). *P<0.05 vs. control group; # P<0.05 vs. STZ group. SO 2 , sulfur dioxide; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; STZ, streptozotocin; HDX, L-Aspartic acid β-hydroxamate.

Article Snippet: The antibodies for matrix metalloproteinase (MMP)9, MMP24, tissue inhibitor of metalloproteinase (TIMP)1 and GAPDH were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Staining, Expressing, Standard Deviation, Control