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: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . LC-MS/MS analysis of protein complex coimmunoprecipitated with TIMP-1 in PANC-1 cells. (I) Coomassie blue staining of 10% SDS-PAGE gel. Red arrowheads indicate protein complexes in which CD63 was detected. Green arrowheads indicate protein complexes in which CD82 was detected. (II) A digested peptide from CD63. (III) A digested peptide from CD82. b . Coimmunoprecipitation of endogenous TIMP-1 with CD63 or CD82 in PANC-1 cells. Cell lysates were immunoprecipitated with rabbit anti–TIMP-1 pAb or control IgG, followed by immunoblotting with mouse anti-CD63 mAb or mouse anti-CD82 mAb. Arrowheads indicate the immunoreactive bands of CD63 or CD82.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining, SDS Page, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . TIMP-1 co-localization with CD82 in cell lines, pearson's coefficient was 0.89±0.13 in PANC-1, 0.46±0.07 in MCF-7, 0.40±0.14 in T13. Scale bar = 20μm. b . In vivo TIMP-1 co-localization with CD82 in DCIS biopsies, pearson's coefficient was 0.26±0.01. Cells were double-immunostained with mouse anti-CD82 mAb and rabbit anti–TIMP-1 pAb. Superimposed areas are in yellow. Normal: healthy controls in the same DCIS biopsy sample. Scale bar = 200μm. c . In vivo TIMP-1 co-localization with CD82 in PDAC biopsies, pearson's coefficient was 0.46±0.18. Cells were double-immunostained with mouse anti-CD82 mAb and rabbit anti–TIMP-1 pAb. Benign: healthy/atypical hyperplasia control in the same PDAC biopsy sample. Scale bar = 200μm.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: In Vivo

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . Bioinformatics analysis of TIMP-1 and CD82-LEL binding. (I) Ribbon representation of TIMP-1 (amino acids 24-204) based on the SWISS-MODEL template 1UEAB. The modeled homolog shares 98.9% similarity with TIMP-1 (amino acids 24-204). (II) CD82-LEL (amino acids 111-228) 3D structure by Phyre. It covers 64% of the LEL with 99.8% confidence, and presented the tetraspanin family characteristic of an α-helix structure before the conserved CCG motif. 16 (III and IV) TIMP-1 and CD82-LEL (III) and TIMP-1–MMP3 and CD82-LEL (IV) top protein–protein docking results. Notably, the original TIMP-1–MMP3 data (PDB accession: 1UEA) contained two copies of the complex. We used Swiss-PdbViewer to simplify it into a single copy. (V and VI) Ligand center of mass positions for the top 500 ZDOCK models corresponding to III and IV, respectively. b . TIMP-1 and CD82-LEL binding by chemical cross-linking experiments. Lanes 1 and 2: CD82-LEL–SBED and TIMP-1–SBED in non-UVA condition, respectively. Lanes 3 and 4: CD82-LEL–SBED and TIMP-1–SBED in UVA condition, respectively. Lane 5: CD82-LEL–SBED mixed with recombinant TIMP-1 followed by UVA exposure. Lane 6: TIMP-1–SBED mixed with recombinant CD82-LEL followed by UVA exposure. Lanes 7 and 8: Same as lanes 5 and 6. Lanes 9 and 10: Same as lanes 7 and 8, but supplemented with DTT. Arrowheads indicate target bands. c . TIMP-1 and CD82-LEL binding by competitive binding analysis. Molar excess CD82-LEL competed with 125I-labeled CD82-LEL to bind to TIMP-1. Data are representative of two independent experiments performed in duplicate. Error bars represent SD . ** p < 0.01. d . Single-molecule force spectroscopy characterization of the binding strength between TIMP-1 and CD63-LEL/CD82-LEL. (I) Experimental scheme. (II) Typical traces obtained from single-molecule force spectroscopy measurement for TIMP-1–CD63-LEL binding. (III) Distribution of the unbinding forces between TIMP-1 and CD63-LEL. (IV) Representative unbinding traces of TIMP-1–CD82-LEL. (V) Distribution of the unbinding forces between TIMP-1 and CD82-LEL.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Binding Assay, Recombinant, Labeling, Spectroscopy

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . Culture system model of 293A cells and PANC-1 cells. b . The culture system was effective. (I) 293A cells were transfected with peGFP-N2 or pTIMP-1–eGFP. Cell lysates were immunoblotted with anti-GFP and anti–α-tubulin mAbs. (II) ELISA of TIMP-1 in 293A culture supernatant. TIMP-1 level in culture supernatant from 293A cells transfected with pTIMP-1-eGFP was almost twice as much as it from 293A cells transfected with eGFP. (III) PANC-1 cells were treated with culture supernatant from eGFP/eGFP-TIMP-1 transfected 293A cells. Outside-in GFP was detectable in PANC-1 cells. Cell lysates were immunoblotted with anti-GFP and anti–α-tubulin mAbs. Endogenous CD82 could be immunoprecipitated by outside-in TIMP-1 (eGFP-tagged) in PANC-1 (as arrowhead pointed at). c . TIMP-1–eGFP membrane–cytoplasm translocation in PANC-1 cells. PANC-1 cells were cultured with culture supernatant of 293A cells transfected with peGFP-N2 or pTIMP-1–eGFP. PANC-1 cells were immunostained with mouse anti-GFP mAb and counterstained with DAPI. d . CD82 mediated TIMP-1–eGFP membrane–cytoplasm translocation in PANC-1 cells. PANC-1 cells which were transfected with siNC (100 nM) or siCD82 (100 nM) for 48h were treated with culture supernatant of 293A cells transfected with peGFP-N2 or pTIMP-1–eGFP. PANC-1 cells were immunostained with mouse anti-GFP mAb and counterstained with DAPI. Scale bar = 20μm. CD82 deletion was confirmed by western blotting. e . CD82-eYFP fusion protein overexpression by pCD82-eYFP transfection. 293A cells were transfected with peYFP-N2 or pCD82-eYFP for 24 h. Cell lysates (using 1% Triton X-100) were immunoblotted with mouse anti-CD82 and anti–α-tubulin mAbs. Images shown are representative of eYFP and CD82-eYFP distribution in live PANC-1 cells. Scale bar = 20μm. f . TIMP-1 protein but not TIMP-2 triggers CD82 leaving from cytomembrane into cytoplasm. Images shown are representative of intense foci of CD82-eYFP fluorescence in the 0 th , 3 th , 15 th minute since TIMP-1 or TIMP-2 protein (100 ng/ml) addition in live PANC-1 cells. The scatter diagram quantified the images in the right by calculating outside-in spots (less than 0.5μm, reflecting CD82) in PANC-1 cytoplasm using Imaris8.0.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Translocation Assay, Cell Culture, Western Blot, Over Expression, Fluorescence

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . Migration inhibitory effect of cyclopamine and TIMP-1 in PANC-1 cells. Cells were treated with cyclopamine (10 μM) or TIMP-1 (50, 100 ng/mL) for 24 h. b . Mild CD82 deletion weakened TIMP-1 inhibition of PANC-1 cell migration. Cells were transfected with siNC (20 nM) or two independent siRNAs (20 nM) targeting CD82 for 48h. Cells were then treated with TIMP-1 (100 ng/mL) for 6 h. Scale bar = 60μm. c . TIMP-2 had no inhibitory effect on PANC-1 cell migration. Cells were treated with TIMP-2 or cyclopamine (10 μM) for 24 h. d . DN–TIMP-1 significantly weakened TIMP-1 inhibition of PANC-1 cell migration. Cells were treated with TIMP-1 (100 ng/mL) or DN–TIMP-1 (100 ng/mL) for 18 h. Error bars represent SD . * p < 0.05; ** p <0.01; *** p <0.001; ns denotes no statistical significance.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Migration, Inhibition, Transfection

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . TIMP-1 modified cytoskeleton remodeling via CD82. Cells were transfected with siNC or siCD82 and treated with TIMP-1 (100 ng/mL) for 18 h. (I) Cells were stained with fluorescent phalloidin, counterstained with DAPI and analyzed. Scale bar = 20μm. (II) Relative intensity of F-actins was qualified by ImageJ. (III) CD82 deletion was confirmed by western blotting. b . TIMP-1 increased intercellular ATP concentration through CD82. Cells were transfected with siNC or siCD82 and went through serum starvation for 6 h. Cell were lysed for ATP detection without treatment or after TIMP-1 (100 ng/mL) stimulation. Error bars represent SD . * p < 0.05; ** p <0.01; *** p <0.001.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Modification, Transfection, Staining, Western Blot, Concentration Assay

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . Immunohistochemistry staining scores of TIMP-1 and CD82 in patients with pancreatic diseases (n=32). b . Cellular location of TIMP-1 and CD82. Photomicrographs represent TIMP-1 (rabbit anti–TIMP-1 pAb) and CD82 (mouse anti-CD82 mAb) immunostaining. Arrowheads delineate duct epithelial cells. Scale bar = 200μm. c . ROC curve was plotted to determine the accuracy of TIMP-1 as diagnostic test to discriminiate between PDAC with metastasis lesions and without metastasis lesions. * p < 0.05; ** p <0.01; ns denotes no statistical significance.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Immunohistochemistry, Staining, Immunostaining, Diagnostic Assay

Journal: Oncotarget

Article Title: TIMP-1 and CD82, a promising combined evaluation marker for PDAC

doi: 10.18632/oncotarget.14133

Figure Lengend Snippet: a . Theoretical model of the TIMP-1 receptor on the cell surface of pancreatic carcinoma cells. After directly binding to TIMP-1, CD82 or CD63 forms a hexameric complex containing five other tetraspanins. Each hexamer comprises a ring of six inner (SELs) and six outer parts (LELs). b . Hypothetical development of PDAC.

Article Snippet: Recombinant human TIMP-1 (Cys 24-Ala 207), TIMP-2 (Cys 27-Pro 220) and CD82-LEL (Gly 111-Leu 228) were procured from Sino Biological, Inc. (Beijing, China).

Techniques: Binding 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