Journal: Nucleic Acids Research

Article Title: Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation

doi: 10.1093/nar/gkaf1290

Figure Lengend Snippet: Dynamic regulation of biosynthesis pathway with tryptophan-responsive MR-scRNAs. ( A ) Design schematic illustrating the dynamic regulatory strategy used to conditionally activate the violacein biosynthesis pathway in response to concentrations of the upstream metabolite, tryptophan, that is required for both growth and biochemical production. ( B ) Dose–response curve of the tryptophan MR-scRNA in response to the target metabolite. ( C ) Violacein titer extracted from E. coli cells with the violacein biosynthesis pathway activated by an on-target scRNA (+), non-targeting scRNA (−), or a tryptophan-responsive scRNA (MR-scRNA). Abbreviations: Ø, not detected; a.u., arbitrary units.

Article Snippet: Violacein biosynthesis genes were obtained from Addgene plasmid 73440 (a gift from Mattheos Koffas) [ ].

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca 2+ -dependent Nrf2 pathway.

doi: 10.1016/j.biopha.2023.115776

Figure Lengend Snippet: Fig. 2. CETSA-LC–MS/MS analysis to identify the protein target of CHP. (A) Workflow of the CETSA-LC–MS/MS method. Live cells are treated with DW (control) or CHP (20 μM) and then heated to 55 ◦C or 60 ◦C. After cell lysis, soluble proteins are digested and peptides are labeled with TMT-6 plex reagent. Peptide desalting, HPLC fractionation, and LC–MS/MS analysis are performed sequentially. (B) Schematic diagram of step-by-step criteria for target selection. (C) Functional analysis of target protein candidates. (D) Candidate target proteins with significantly increased thermal stability upon binding to CHP. Black circle: 28 proteins whose stability was increased by more than 50% (FC≥1.5) compared to the increase at 55 ◦C. Red circle: CLIC1. (E) The raw intensity value of CLIC1 peptide was converted to a log value.

Article Snippet: A CLIC1 (Myc-DDKtagged) human ORF clone (OriGene, RC218042) was used to generate CLIC1 mutant vectors.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Lysis, Labeling, Fractionation, Selection, Functional Assay, Binding Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca 2+ -dependent Nrf2 pathway.

doi: 10.1016/j.biopha.2023.115776

Figure Lengend Snippet: Fig. 3. Immunoblot analysis of target candidate from the CETSA-LC–MS/MS analysis. (A) Immunoblot results evaluating the thermal stability of CLIC1 in HEK293 after treatment with CHP (20 μM). (B) The binding affinity of CHP and CLIC1 at 60 ◦C as a function of CHP dose in HEK293 cells determined by isothermal CETSA. (C) A graph of the thermal stabilization curve according to Western blot quantification and EC50 values. (D) CETSA assay was conducted to confirm the binding of CHP to CLIC1 in LX-2 cells.

Article Snippet: A CLIC1 (Myc-DDKtagged) human ORF clone (OriGene, RC218042) was used to generate CLIC1 mutant vectors.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca 2+ -dependent Nrf2 pathway.

doi: 10.1016/j.biopha.2023.115776

Figure Lengend Snippet: Fig. 4. Analysis of the binding site of CHP for CLIC1. (A) To analyze the interaction of CHP and CLIC1, molecular docking analysis was performed using Discovery Studio software. CDOCKER E: − 11.9712 kcal/mol. (B) Two-dimensional figures showing the amino acid residues of possible binding sites for CHP and CLIC1. (C) For CETSA, HEK293 cells were transfected with MYC-CLIC1WT, MYC-CLIC1E228Q, or MYC-CLIC1A232E vectors for 48 h. The cells were then harvested and treated with CHP. After heat treatment for 3 min, protein lysates were obtained. The E228Q mutation abolished the CHP-mediated protection of CLIC1 from thermal degradation. (D) Cellular ROS levels were measured in HEK293 cells transfected with MYC-CLIC1WT, MYC-CLIC1E228Q, or MYC-CLIC1A232E treated with CHP (20 μM) for 24 h after pretreatment with TGF-β (2 ng/mL). *p < 0.05, * *p < 0.01, * **p < 0.001, ns: no significant difference.

Article Snippet: A CLIC1 (Myc-DDKtagged) human ORF clone (OriGene, RC218042) was used to generate CLIC1 mutant vectors.

Techniques: Binding Assay, Software, Transfection, Mutagenesis

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca 2+ -dependent Nrf2 pathway.

doi: 10.1016/j.biopha.2023.115776

Figure Lengend Snippet: Fig. 5. CLIC1 as a biologically relevant protein target of CHP. (A) Cytosolic calcium levels were reduced after CLIC1 knockdown for 24 h using siCLIC1 (50 nM) (scale bar: 50 µm). (B) CLIC1 knockdown increased the phosphorylation of Nrf2 and affected the downstream factor HO-1. (C) CLIC1 knockdown reduced COL1A1, FN, α-SMA levels at later time points (48 h). (D) Pretreatment with Vera (verapamil, 10 μM) for 30 min followed by treatment with CHP (20 μM) or IAA-94 (30 μM) for 4 h inhibited the increase in cytosolic calcium (scale bar: 50 µm). *p < 0.05, * *p < 0.01, * **p < 0.001, * ** *p < 0.0001.

Article Snippet: A CLIC1 (Myc-DDKtagged) human ORF clone (OriGene, RC218042) was used to generate CLIC1 mutant vectors.

Techniques: Knockdown, Phospho-proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca 2+ -dependent Nrf2 pathway.

doi: 10.1016/j.biopha.2023.115776

Figure Lengend Snippet: Fig. 6. Role of CLIC1 in CCl4-induced liver fibrosis in mice. (A) Experimental design of the in vivo study. (B) Representative images of liver sections stained with H&E, Sirius red, and immunohistochemistry for fibronectin and collagen IV in fibrotic liver tissue of CCl4-treated mice. (Scale bar: 100 µm) (C) Liver hydroxyproline concentration in WT or CLIC1 KO mice. (D) Gene expression levels of the inflammatory cytokine IL-6 of in the liver of WT or CLIC1 KO mice. (E-F) Protein expression levels of ECM fibrotic marker in mouse liver. (G) Nrf2 protein expression levels in mouse liver. (H) HO-1 mRNA expression levels in mouse liver. *p < 0.05, * *p < 0.01, * **p < 0.001, n.s: no significant difference.

Article Snippet: A CLIC1 (Myc-DDKtagged) human ORF clone (OriGene, RC218042) was used to generate CLIC1 mutant vectors.

Techniques: In Vivo, Staining, Immunohistochemistry, Concentration Assay, Gene Expression, Expressing, Marker

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca 2+ -dependent Nrf2 pathway.

doi: 10.1016/j.biopha.2023.115776

Figure Lengend Snippet: Fig. 7. Schematic illustration of the mechanism of antifibrotic action of the cyclic dipeptide. CHP increases intracellular calcium levels by targeting CLIC1. Sub sequently, by activating the Nrf2 pathway, the expression of antioxidant genes is induced, and ROS levels are reduced, thereby decreasing the ECM levels. The finding that CLIC1 is responsible for the antioxidant activities of CHP suggests a novel role for CLIC1 in fibrosis.

Article Snippet: A CLIC1 (Myc-DDKtagged) human ORF clone (OriGene, RC218042) was used to generate CLIC1 mutant vectors.

Techniques: Expressing