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: 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