Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 2 Loci-specific transactivation of the mouse TIMP promoters using dCas9.nVP64. Notes: Fold-induction over the no gRNA transfection controls using dCas9.nVP64 (black circle) or dCas9 (orange triangle) and the relative gRNAs targeting loci within the (A) TIMP1, (B) TIMP2, or (C) TIMP3 core promoters. Closed symbols represent targeting of the top strand (also indicated by T) while open symbols represent targeting of the bottom strand (indicated by B) of the promoter DNA. X-axis shows the relative distance from the start codon and the numerous transcription factor-binding sites located within the mouse TIMP promoters. Data from Clark et al.24 Mean±standard error of the mean , n=3. Abbreviation: TIMP, tissue inhibitor of metalloproteinases.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Transfection, Binding Assay

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 3 Comparison of the transactivation efficiency of TIMP1 and TIMP2 promoters using different transcriptional activating effectors. Notes: (A) Illustration of the different dCas9 constructs used. (B, C) Fold luciferase induction over no gRNA transfection controls comparing VP64-, VP160-, or VPR. dCas9 effector constructs targeting 3 loci within the mouse TIMP1 (B) or 4 loci within the mouse TIMP2 (C) promoter. Mean±standard error of the mean (SEM), *P<0.025, **P<0.005, ***P<0.0005, ****P<0.0001, unpaired t-test, n=4. (D, E) Fold luciferase induction over no gRNA transfection comparing VPR targeting 4 loci in the mouse TIMP1 (D) and mouse TIMP2 (E) promoter to SAM targeting between 0 and 250 bps upstream of the transcription start site. Mean±SEM, **P<0.01, ***P<0.005, one-way analysis of variance with multiple comparisons, n=4. Abbreviations: CMV, cytomegolovirus; eGFP, enhanced green fluorescent protein; HA, hemagglutinin; NLS, nuclear localization sequence; SAM, synthetic activation motif; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Comparison, Construct, Luciferase, Transfection, Sequencing, Activation Assay, Virus

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 4 Endogenous TIMP gene activation in mouse NSC34 cells. Notes: (A) PMA stimulation enhances promoter-reporter activity for TIMP1 and 3. TIMP promoter-reporter constructs were transfected into HEK293 cells. The promoter activity (luciferase) was assessed by the Dual-Glo assay 12 hours after DMSO or PMA stimulation at the indicated concentrations. Mean±standard error of the mean (SEM), *P<0.05, **P<0.01 or n.s., n=3. (B) Endogenous TIMP mRNA basal expression in unstimulated NSC34 cells relative to GAPDH. Mean±SEM, n=6–11. All bars were significantly different (P<0,001) from each other by a pair-wise two-tailed t-test. Specific endogenous Cas9-VPR mediated transactivation using gRNAs targeting the mouse TIMP1 (C) and mouse TIMP2 (D) promoters. Dashed bar indicates the expression of all 4 gRNAs simultaneously. Mean±SEM, *P<0.05, **P<0.01, one-way analysis of variance with multiple comparisons, n=4. The “All” condition was analyzed separately; mean±SEM, ****P<0.0001, unpaired t-test, n=4. Abbreviations: DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; n.s., not significant; PMA, phorbol-12-myristate-13-acetate; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Activation Assay, Activity Assay, Construct, Transfection, Luciferase, Glo Assay, Expressing, Two Tailed Test

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 5 Production of functional TIMP2 protein by gRNA-induced NSC34 cells. Notes: (A) Reverse zymography of cell culture supernatant. The lanes were loaded with control recombinant TIMP1 or 2 (10, 3,1, or 0 ng), or cell culture supernatant from NSC34 cells transfected with nothing, dCas-VPR alone, dCas- VPR with a negative control blank gRNA, or dCas-VPR with all 4 (p-194, p-535p, p-610, and p-956) TIMP2 gRNAs. The transfection condition was identical to that for mRNA assay except that the culture supernatant was harvested 48 hours after transfection. The blot is representative of 3 biological replicates. (B) Time course of increase in fluorescence due to cleavage of the fluorogenic MMP substrate. The slope of the fluorescence vs time plot was taken as a measure of the relative catalytic activity. The plot is representative of 3 biological replicate experiments. (C) A bar plot of relative MMP9 catalytic activity (mean±standard error of the mean, n=3). Note significant inhibition (****P<0.0001) of the catalytic activity by cell culture supernatant from wells transfected with all 4 TIMP2 gRNAs. Abbreviations: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Functional Assay, Zymography, Cell Culture, Control, Recombinant, Transfection, Negative Control, Fluorescence, Activity Assay, Inhibition

Journal: Cell metabolism

Article Title: Inhibition of acetyl-CoA carboxylase (ACC) by phosphorylation or by the liver-specific inhibitor, ND-654, suppresses lipogenesis and hepatocellular carcinoma

doi: 10.1016/j.cmet.2018.08.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TaqMan Assay IDs are as follows: for rat studies: 18S : Hs03003631_g1; Cxcl1 : Rn00578225_m1; Il-1b : Rn00580432_m1; Il-6 : Rn01410330_m1; Tnfa : Rn99999017_m1; Cd80 : Rn00709368; and Cd163 : Rn01492519_m1, and for mouse studies: Tbp : Mm01277042_m1; Il-6 Mm00446190_m1; Tnfa Mm00443258_m1; Timpl : Mm01341361_m1; Col1a1 : Mm00801666_g1; Co14a1 : Mm01210125_m1; Acata2 : Mm00725412_s1.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: TIMP-1 Attenuates the Development of Inflammatory Pain Through MMP-Dependent and Receptor-Mediated Cell Signaling Mechanisms

doi: 10.3389/fnmol.2019.00220

Figure Lengend Snippet: Assessing TIMP-1 expression along peripheral nociceptive circuit following cutaneous inflammation. (A) Cutaneous inflammation does not alter overall TIMP-1 protein expression in lumbar spinal cord or (B) DRG, but does increase protein expression in panel (C) hairy skin. n = 4/condition, ∗ indicate significant differences compared to naïve controls, p < 0.05, and error bars depict SEM.

Article Snippet: WT and T1KO mice received injections (s.c.) of recombinant murine (rm)TIMP-1 (10 ng/μL, 10 μL; R&D Systems; Minneapolis, MN) immediately following CFA injection (10 μL) into the right hindpaw.

Techniques: Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: TIMP-1 Attenuates the Development of Inflammatory Pain Through MMP-Dependent and Receptor-Mediated Cell Signaling Mechanisms

doi: 10.3389/fnmol.2019.00220

Figure Lengend Snippet: Cellular colocalization of TIMP-1 expression. (A) Immunostaining (20×) of naïve and inflamed lumbar spinal cord 24 h following inflammation. TIMP-1 (green) expression is localized to GFAP-positive astrocytes (red). n = 3/condition, scale bar 20 μm. (B) Immunostaining (20×) of naïve and inflamed lumbar DRG 24 h following inflammation. TIMP-1 (green) expression is colocalized with by GFAP-positive satellite glial cells (red). n = 3/condition, scale bar 20 μm. (C) Immunostaining (40×) of hindpaw hairy skin shows K14-positive keratinocytes (red) upregulate TIMP-1 (green) 24 h following inflammation compared to naïve control. n = 3/condition, scale bar 50 μm.

Article Snippet: WT and T1KO mice received injections (s.c.) of recombinant murine (rm)TIMP-1 (10 ng/μL, 10 μL; R&D Systems; Minneapolis, MN) immediately following CFA injection (10 μL) into the right hindpaw.

Techniques: Expressing, Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: TIMP-1 Attenuates the Development of Inflammatory Pain Through MMP-Dependent and Receptor-Mediated Cell Signaling Mechanisms

doi: 10.3389/fnmol.2019.00220

Figure Lengend Snippet: Mice lacking TIMP-1 develop thermal and mechanical hypersensitivity following cutaneous inflammation. (A) No differences in baseline thermal PWL are exhibited between T1KO and WT mice ( n = 16/condition). (B) Inflamed T1KO mice exhibit significantly reduced PWLs compared to inflamed WT mice and naïve WT and T1KO mice ( n = 8/condition). (C) Baseline assessment of mechanical PWTs revealed no genotypic differences between T1KO ( n = 20) and WT mice ( n = 18). (D) T1KO mice develop significantly reduced PWTs 1, 5, and 7 days following CFA administration compared to WT controls ( n = 8–10/condition). ∗ = signficantly different from mice in all other conditions, p < 0.05, and error bars depict SEM.

Article Snippet: WT and T1KO mice received injections (s.c.) of recombinant murine (rm)TIMP-1 (10 ng/μL, 10 μL; R&D Systems; Minneapolis, MN) immediately following CFA injection (10 μL) into the right hindpaw.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: TIMP-1 Attenuates the Development of Inflammatory Pain Through MMP-Dependent and Receptor-Mediated Cell Signaling Mechanisms

doi: 10.3389/fnmol.2019.00220

Figure Lengend Snippet: Mice lacking TIMP-1 show increased sensitivity in non-inflamed tissues. (A) Injection of CFA into the hairy skin causes mechanical hypersensitivity on the plantar surface of the paw to develop 1 day following inflammation in T1KO, but not WT, mice. (B) Graph depicting mechanical responsiveness following inflammation collapsed across time. Inflamed T1KO mice greater mechanical sensitivity overall following cutaneous inflammation. (C) Administration of TIMP-1(FL), TIMP-1(N), or TIMP-1(C) into the hairy skin at the time of inflammation prevents the development of mechanical hypersensitivity in T1KO mice. (D) Hindpaw administration of CFA produces mechanical hypersensitivity on the paw contralateral to inflammation in T1KO relative txo WT mice. Treatment with rmTIMP-1 attenuated contralateral hypersensitivity in T1KO mice. PWT are presented as change from baseline. n = 8/condition, ∗ represent significant differences relative to naïve controls, p < 0.05, and error bars depict SEM. # significantly different from WT mice.

Article Snippet: WT and T1KO mice received injections (s.c.) of recombinant murine (rm)TIMP-1 (10 ng/μL, 10 μL; R&D Systems; Minneapolis, MN) immediately following CFA injection (10 μL) into the right hindpaw.

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: TIMP-1 Attenuates the Development of Inflammatory Pain Through MMP-Dependent and Receptor-Mediated Cell Signaling Mechanisms

doi: 10.3389/fnmol.2019.00220

Figure Lengend Snippet: Replacement of TIMP-1 attenuates ongoing inflammatory pain in WT mice. Comparison of pre-conditioning and post-conditioning time spent in the clonidine paired chamber show a significant increase in the post-conditioning time in the Vehicle/CFA treated mice, but not the rTIMP-1/CFA treated mice. ∗ p < 0.01 vs. pre-conditioning time. Sample size CFA/rTIMP-1 = 9; CFA/Veh = 8. Error bars = S.E.M.

Article Snippet: WT and T1KO mice received injections (s.c.) of recombinant murine (rm)TIMP-1 (10 ng/μL, 10 μL; R&D Systems; Minneapolis, MN) immediately following CFA injection (10 μL) into the right hindpaw.

Techniques: Comparison