recombinant human timp3  (R&D Systems)

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 3. Transcriptional and protein expression of Timp3. (A) Schematic illustration of TIMP3′s inhibitory activity on matrix metalloproteinases (MMPs) and on disintegrin and metalloprotease 17 (ADAM17), also called TACE (tumor necrosis factor-α-converting enzyme). (B, C) RNAscope localization of Timp3, Mmp14 and Adam17 mRNA expression in DRG of Plp1-Cre/tdTomato mice, wherein Cre recombinase is expressed in satellite glial cells (SGCs) (B), and in naïve CD1 mice (C). Arrowheads indicate mRNA colocalization of Mmp14 and Adam17 with Timp3 in SGCs, * indicates neurons. Scale bars = 25 μm in (B) and 5 μm in (C). (D) PCR in mouse and human DRG tissues. Samples with omitted RT (reverse transcriptase) show no bands, confirming the specificity of the amplification. (E) Immunofluo rescence of TIMP3 in human DRG tissue. Scale bars = 50 μm. DAPI was used as counterstain. # indicates the fluorescent signal due to the presence of lipofuscins in human DRG neurons.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Expressing, Activity Assay, RNAscope, Reverse Transcription, Amplification

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 4. Timp3 controls mechanical and thermal sensitivities in naïve mice. (A) Schematic illustration of the experiment showing the timeline of siRNA injections, pharmacological and biochemical studies. (B) Western blot representative image and quantification show that Timp3 siRNA significantly decreases Timp3 protein levels in DRGs tissues (n = 4). (C) Timp3 siRNA injections do not cause locomotor dysfunction in the Rota-rod test (n = 5). (D) Mechanical and thermal (von Frey, Hargreaves, and dry ice) allodynia induced by Timp3 siRNA compared to a control (Ctrl) non-targeting siRNA (2 µg of siRNA per delivery in the transfection agent PEI, n = 7). (E) Anti-allodynic effect of exogenous recombinant TIMP3 (rTIMP3, 100 ng/site, i.t.), general endogenous tissue inhibitor of MMPs (TIMP-1, 4 pmol/ site), MMP2 and MMP14 inhibitors (10 µg/site, i.t.), TACE/ADAM17 inhibitor (TAPI-2, 1 µg/site, i.t.), and a neutralizing antibody for TNF-α (5 µg/site, i.t.) on mechanical allodynia induced by Timp3 siRNA on day 2. (F) Anti-TIMP3 antibody (TIMP3 Ab, 10 µg/site, i.t.) induces mechanical allodynia compared to IgG control in male and female mice. BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, E) and Two-way ANOVA followed by Sidak’s post hoc test (D, F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Western Blot, Control, Transfection, Recombinant, Two Tailed Test

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 5. Recombinant TIMP3 protein reverses and prevents mechanical and cold allodynia in a mouse model of chemotherapy-induced neuropathic pain. (A) Schematic of the experiment showing the timeline of paclitaxel (PAX) or vehicle injections, pharmacological studies, and immunohistochemistry (IHC) analysis. (B) Representative image and (C) quantification of Timp3 protein in mouse DRG tissue 14 days after first injection of PAX or vehicle control (n = 5). (D, E) Paclitaxel- induced mechanical (von Frey) and (F) cold allodynia (dry ice) are significantly, dose-dependently reversed up to 6 h by single intrathecal administration of re combinant TIMP3 protein (rTIMP3, 3–100 ng/site) delivered at day 14 after chemotherapy injection (n = 4–5). (G) Schematic of experiment showing timeline of PAX injections concomitantly with rTIMP3 or PBS, behavioral tests, transcriptional, and histological analysis. (H, I) Repeated administrations of rTIMP3 (100 ng/site, i.t.) prevent paclitaxel-induced mechanical and cold allodynia (n = 5–6). BL = baseline. Data expressed as mean ± SEM, statistically analyzed by two-tailed t-test (C), two-way ANOVA followed by Sidak’s post hoc test (D, F, H, I), and one-way ANOVA followed by Tukey’s post hoc test (E). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Recombinant, Immunohistochemistry, Injection, Control, Two Tailed Test

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 6. Transcriptional analyses of TIMP3 signaling in cultured SGCs after paclitaxel treatment. (A) Schematic of the experi mental design used in cultured SGCs. (B) Representative image and quantification of immunofluorescence intensity of GFAP protein in SGC culture after 24 h of incu bation with paclitaxel (PAX, 300 nM) or vehicle control (Veh; n = 4). (C) Quantifi cation of SGC culture viability 24 h after PAX or Veh treatment (n = 6). (D) Quan tification of mRNA expression levels of Timp3, Mmp2, Mmp14 and Adam17 in SGC culture after PAX or Veh incubation (n = 6). (E) Heat map of mRNA expression of SGC and metalloprotease signaling markers in SGC culture after incubation with pro saptide Tx14 (1 µM) or pioglitazone (PGZ, 10 µM) and PAX compared to vehicle (n = 3). (F) Schematic illustrating the timeline of Tx14, PGZ, or PBS concomitantly treated with PAX, and the behavioral assay. Repeated injections of (G) Tx14 (10 µg/ site, i.t.) or (H) PGZ (100 µg/site, i.t.) prevent paclitaxel-induced mechanical allodynia (n = 6). BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, D), and Two-way ANOVA followed by Sidak’s post hoc test (G, H): *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Cell Culture, Immunofluorescence, Control, Expressing, Incubation, Behavioral Assay, Two Tailed Test

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: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a TIMP3 mRNA levels of oral cancer tissues and corresponding normal tissues. The TIMP3 mRNA levels of corresponding normal tissues were set as 1. Vertical bars, paired samples. N, corresponding normal tissue. T, oral cancer tissue. b TIMP3 protein levels of oral cancer tissues and corresponding normal tissues, β-actin was used as loading control. N, corresponding normal tissue. T, oral cancer tissue. c TIMP3 protein and mRNA levels of oral cell lines, β-actin and GAPDH was used as loading control. d TIMP3 mRNA levels of oral cell lines were detected by real-time PCR, GAPDH was used as internal control. e TIMP3 methylation levels of CpG island in HNSCC tissues and normal tissues from MethHC database. f The correlation between TIMP3 methylation levels of CpG island and TIMP3 mRNA levels from MethHC database. g TIMP3 mRNA levels of oral cancer cell lines after treatment of 5-aza. h Overview of the TIMP3 CpG locations. Three sequences a (CpG sites: 1–9), b (CpG sites: 10–13), and c (CpG sites: 14–26) were analyzed by pyrosequencing after amplifying P1 and P2 fragments. i The average methylation levels of a, b, and c sequences in oral cell lines. j The correlation between average methylation levels of three sequences and TIMP3 mRNA levels in oral cell lines

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Control, Real-time Polymerase Chain Reaction, Methylation

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Western blot results of Sp1 and TIMP3 after transfection of GFP vector and GFP-SP1 vector, β-actin was used as internal control. b Western blot results of Sp1 and TIMP3 after knockdown of Sp1, β-actin was used as internal control. c TIMP3 promoter activity after transfection of Sp1 overexpression vector, β-gal was used to normalize transfection efficiency. * p < 0.05 compared with GFP. d TIMP3 promoter activity after mutation of the Sp1 binding sites, β-gal was used to normalize transfection efficiency. * p < 0.05 compared with pGL3-TIMP3. e SCC9 and TW2.6 cells were transfected with the control siRNA or Sp1 siRNA. After 24 h, cells were treated with the vehicle control (DMSO) or 5-aza (10 μM) for 96 h before total RNA was subjected to qPCR analysis. * p < 0.05 compared with treatment of scrambled siRNA and DMSO. # p < 0.05 compared with treatment of Sp1 siRNA and DMSO. f SCC9 and TW2.6 cells were treated with the vehicle control (DMSO) or 5-aza (10 μM) for 96 h and were subjected to immunoprecipitation with an antibody against Sp1, DNMT1, and DNMT3B. The precipitates were subjected to PCR amplification using primers directed to Sp1 binding site of the TIMP3 promoter. g DNMT1 and DNMT3B levels in HNSCC tissues and normal tissues from TCGA database. h DNMT1 and DNMT3B levels in oral cancer cell lines and normal oral cell lines. i The mRNA expression of DNMT1 and DNMT3B after transfection of DNMT1 siRNA or DNMT3B siRNA. * p < 0.05 compared with scrambled siRNA. j TIMP3 levels after knockdown of DNMT1 or DNMT3B in SCC9 and TW2.6 cells. * p < 0.05 compared with scrambled siRNA

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Knockdown, Activity Assay, Over Expression, Mutagenesis, Binding Assay, Immunoprecipitation, Amplification, Expressing

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Western blot of SCC9 and TW2.6 stable clones, β-actin was used as internal control. b Cell proliferation was analyzed by MTT assay. The cell number in first day was set as 1 and used for normalization. c Clones were wounded for 0 h, 24 h, and 48 h (SCC9) or 0 h, 3 h, and 6 h (TW2.6). Phase-contrast pictures of the wounds at three different locations were taken. d Migration and invasion abilities were measured after 24 h and 48 h. * p < 0.05 compared with control group. e Migration and invasion abilities of SCC9 and TW2.6 cells exposed to their own stable conditioned medium (CM−: control cells; CM+: SCC9-T9 or TW2.6-T18) as chemoattractant, or to f recombinant TIMP3 protein (rTIMP3). g Migration and invasion abilities of SCC9-T9 and TW2.6-T18 after transfection with scrambled siRNA or TIMP3 siRNA. * p < 0.05 compared with scrambled siRNA

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Western Blot, Clone Assay, Control, MTT Assay, Migration, Recombinant, Transfection

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Morphology and cell size of SCC9 and TW2.6 stable clones. * p < 0.05 compared with control cells. b Adhesion assays of oral stable cells were performed by seeding cells for 30 min on plates coated with collagen. * p < 0.05 com p ared with control cells. c Heat map including 84 EMT-related genes in SCC9-control and SCC9-T9 cells was assessed by Human OneArray ® . Red arrows indicate the downregulation of fibronectin (FN1) and upregulation of E-cadherin (CDH1) in TIMP3 overexpression SCC9-T9 cells. d EMT markers of stable clones were analyzed by real-time PCR. The relative mRNA expression was normalized to GAPDH. * p < 0.05 compared with the control cells. e Western blot of EMT-related protein expression. β-actin was used as loading control. f Western blot of EMT-related protein expression after transfection of scrambled siRNA or TIMP3 siRNA. β-actin was used as loading control. g Knockdown of E-cadherin by siRNA. h Adhesion ability of TIMP3 stable cells after E-cadherin knockdown for 2 days. i Migration ability of TIMP3 stable cells after E-cadherin knockdown for 2 days

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Clone Assay, Control, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Knockdown, Migration

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a The E-cadherin promoter contains three E-box sites was cloned into the luciferase reporter vector. b The E-cadherin promoter activity of SCC9 and TW stable cells after transfection of scrambled siRNA or TIMP3 siRNA. * p < 0.05 compared to control stable cell treated with scrambled siRNA. # p < 0.05 com p ared to TIMP3 stable cell treated with scrambled siRNA. c The mRNA levels of EMT-related transcription factors in TIMP3 stable clones. The relative mRNA expression was normalized to GAPDH. * p < 0.05 compared with the control cells. d The mRNA levels of EMT-related transcription factors of TIMP3 stable cells after transfection with scrambled siRNA or TIMP3 siRNA. The relative mRNA expression was normalized to GAPDH. * p < 0.05 compared with the scrambled siRNA. e The protein expression of EMT-related transcription factors. β-actin was used as loading control. f The protein expression of EMT-related transcription factors after transfection with scrambled siRNA or TIMP3 siRNA. β-actin was used as loading control

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Control, Stable Transfection, Expressing

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a The protein expression of EMT-related signaling pathway in oral stable cells. β-actin was used as loading control. b The protein expression of EMT-related signaling pathway in TIMP3 stable cells after transfection of scrambled siRNA or TIMP3 siRNA. β-actin was used as loading control. c EMT-related protein expression after treatment of PD98059 for 48 h. β-actin was used as loading control. d Migration and invasion abilities after treatment of PD98059 for 48 h. * p < 0.05 compared with DMSO. e EMT-related protein expression after transfection of TIMP3 siRNA for 24 h and treatment of PD98059 for another 24 h. β-actin was used as loading control. f Migration and invasion abilities after transfection of TIMP3 siRNA for 24 h and treatment of PD98059 for another 24 h. * p < 0.05 compared to treatment with TIMP3 siRNA and DMSO

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Expressing, Control, Transfection, Migration

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Luciferase activity image of mice after injecting with luciferase-tagged TW2.6/pcDNA3 or TW2.6/TIMP3 cells. b After 35 days of tumor cell injection, tumors from six mice injected with TW2.6/pcDNA3 or TW2.6/TIMP3 were quantified by measuring the photon influx. c , d Lymph node metastasis was imaged at the end of the study with the mean signal for each group indicated ( n = 6). * p < 0.05 compared with the TW2.6/pcDNA3 groups. e , f Macroscopic analysis of neck lymph nodes. The appearance, number, and volume of neck lymph nodes were photographed, enumerated, and measured after removal. * p < 0.05 compared with the TW2.6/pcDNA3 groups. g Proposed model for the role of TIMP3 methylation contributes to oral cancer metastasis

Article Snippet: The recombinant TIMP3 protein (R&D Systems, Minneapolis, MN, USA) was used at 50 nM.

Techniques: Luciferase, Activity Assay, Injection, Methylation