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: Journal of Extracellular Vesicles

Article Title: CD73 + extracellular vesicles inhibit angiogenesis through adenosine A 2B receptor signalling

doi: 10.1080/20013078.2020.1757900

Figure Lengend Snippet: TIMP-1 carried by pEVs affects tube formation, but not endothelial migration in vitro . (a) Western Blot and relative quantification showing TIMP-1 overexpression in pEVs. Unpaired T test; * P < 0.05. (b) Analysis of the relative tube lengths in the tube formation assay. The treatment of SVEC4-10 with the anti-TIMP-1 5 µg/mL (AF980 R&D) rescued the tube formation. (c) Conversely, the analysis of relative migration in the scratch assay in the presence of the same anti-TIMP-1 antibody did not shown any restoration of the endothelial migration ability in the presence of pEVs. Data are expressed as means ± SEM (3 independent experiments). Kruskal–Wallis test with Dunn’s multiple comparisons test; * P < 0.05.

Article Snippet: The treatment of SVEC4-10 with the anti-TIMP-1 5 µg/mL (AF980 R&D) rescued the tube formation. (c) Conversely, the analysis of relative migration in the scratch assay in the presence of the same anti-TIMP-1 antibody did not shown any restoration of the endothelial migration ability in the presence of pEVs.

Techniques: Migration, In Vitro, Western Blot, Over Expression, Tube Formation Assay, Wound Healing Assay