anxa1 mimetic peptide ac2  (Tocris)

Journal: iScience

Article Title: A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair

doi: 10.1016/j.isci.2023.108565

Figure Lengend Snippet: Identifying potential targets for PGE2 that promote corneal epithelial repair following alkali burns (A) The Venn diagram of the significantly upregulated DEGs between the intestinal organoids (1 μM PGE2 vs. control) and the wounded corneas (wounded cornea vs. control). The transcriptome data was downloaded from the GEO database: GSE116936 and the SRA database with the BioProject access number PRJNA669218 ), respectively. Overlapped DGEs were plotted in a heatmap. (B) The expression and distribution of the 52 overlapped DGEs in corneas. Upper: heatmap showing the 52 overlapped DGEs in alkali-burned corneas; Lower: dot plot showing the gene expression level and distribution in different corneal cells. The single-cell transcriptome data was downloaded from the GEO database: GSE186433 . (C) Gene expression analysis of Anxa1 , Il1b , Il6 , and Ccl2 in mice corneas at different time post alkali burns (n = 3). Values are normalized to GAPDH and displayed relative to WT. Error bars indicate mean±SEM.

Article Snippet: ANXA1 mimetic peptide Ac2-26 was purchased from Tocris Bioscience Company (Bristol, UK) and dissolved in PBS to make a 10 ng/μL concentration.

Techniques: Control, Expressing, Gene Expression

Journal: iScience

Article Title: A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair

doi: 10.1016/j.isci.2023.108565

Figure Lengend Snippet: The effect of ANXA1 on the corneal epithelial repair after alkali burns and the ANXA1 expression response to different dosages of PGE2 (A) Gene expression analysis of ANXA1 in hCECs after treatment with 10526 (n = 3). Values are normalized to GAPDH and displayed relative to the DMSO vehicle. (B and C) Gene expression analysis of ANXA1 in hCECs after treatment with different dosages of PGE2 (B) and dmPGE2 (C) (n = 3). Ordinary one-way ANOVA was used, p < 0.0001 in B and p = 0.0052 in C. (D and E) Representative images (D) and statistical analysis (E) of the wound healing of the scratch in hCECs with the overexpression of ANXA1 (n = 3). Scale bar: 400 μm. (F and G) Representative images of fluorescein sodium staining (F) and statistical analysis (G) of corneal epithelial wound healing after alkali burn with the treatment of a mimetic peptide of ANXA1 (Ac2-26) (n = 4). 0.1% DMSO was used as the DMSO vehicle control, and PBS was used as the PBS vehicle control. Error bars indicate mean±SEM. ∗p < 0.05, and ∗∗p < 0.01.

Article Snippet: ANXA1 mimetic peptide Ac2-26 was purchased from Tocris Bioscience Company (Bristol, UK) and dissolved in PBS to make a 10 ng/μL concentration.

Techniques: Expressing, Gene Expression, Over Expression, Staining, Control

Journal: iScience

Article Title: A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair

doi: 10.1016/j.isci.2023.108565

Figure Lengend Snippet: Screening the target elements contributing to a high dosage of PGE2 inhibiting the activity of the ANXA1 promoter (A) The analysis of different dosages of PGE2 on the activity of the ANXA1 promoter. The 2000 bp ANXA1 promoter was used as the full-length promoter for analysis by dual luciferase activity assay (n = 4). (B) Effects of a moderate dosage and a high dosage of PGE2 on the activity of the ANXA1 promoters with different lengths. The 2000 bp ANXA1 promoter was shortened to four lengths with an interval of 500 bp (n = 3). (C) Effects of a high dosage of PGE2 on the activity of the ANXA1 promoter with different lengths. The 1000 bp ANXA1 promoter was shortened to 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, and 1000 bp with an interval of 100 bp (n = 3). (D) Schematic illustration of the predicted cis regulate elements (CREs) located in -700—-800 bp and -900—-1000 bp of the ANXA1 promoter. "+1" represents the transcription start site. (E) The ANXA1 expression analysis after the overexpression of GATA3 in hCECs (n = 3). qPCR and western blot were used. Values are normalized to GAPDH and β-actin and displayed relative to the empty vector control, respectively. (F) Effects of GATA3 on the luciferase activity of the 1000 bp ANXA1 promoter with the predicted GATA3 CRE1 and CRE2 mutation (n = 3). The activity of ANXA1 promoter with the mutation of GATA3 CRE1 and CRE2 was compared with the control ANXA1 promoter. #p < 0.05, ##p < 0.01. The activity of ANXA1 promoter with the overexpression of GATA3 was compared with the corresponding control group. 0.1% DMSO was used as the DMSO vehicle control. Error bars indicate mean±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p<0.001, ∗∗∗∗p < 0.0001, and ns, no significance.

Article Snippet: ANXA1 mimetic peptide Ac2-26 was purchased from Tocris Bioscience Company (Bristol, UK) and dissolved in PBS to make a 10 ng/μL concentration.

Techniques: Activity Assay, Luciferase, Expressing, Over Expression, Western Blot, Plasmid Preparation, Control, Mutagenesis

Journal: iScience

Article Title: A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair

doi: 10.1016/j.isci.2023.108565

Figure Lengend Snippet: A high dosage of PGE2 inhibited ANXA1 expression by enhancing GATA3 expression and reducing CREB1 phosphorylation (A) Effects of a high dosage of PGE2 on the activity of the 1000 bp ANXA1 promoter with the mutation of predicted GATA3 CRE1 and CRE2 (n = 3). (B) Effects of a high dosage of PGE2 on the activity of the 1000 bp ANXA1 promoter with the mutation of predicted GATA3 CRE2 and CREB1 CRE. The arrows and diamonds filled with white indicate the corresponding CRE mutations. (C) Analysis of GATA3 expression and phosphorylation in hCECs after treatment with different dosages of PGE2. (D) Analysis of CREB1 phosphorylation in hCECs after treatment with different dosages of PGE2. (E) The binding analysis of the nucleoproteins of hCECs with the GATA3 CRE2 and CREB1 CRE probe by electrophoretic mobility shift assays. GATA3 CRE2 and CREB1 CRE probe concentrations were 1 μM; nucleoprotein proteins were 2.5μg and 12.5 μg, respectively; the concentrations of competing probes were 100 nM, 250 nM, 2.5 μM, and 25 μM. (F) The binding analysis of the nucleoproteins of hCECs with the probes after co-incubation with GATA3 antibody. The probe concentration was 1 μM; Nucleoprotein protein was 10 μg; antibody was 1 μg, respectively. (G) The binding analysis of the nucleoproteins of hCECs after treatment with different dosages of PGE2 with probes by electrophoretic mobility shift assays. The probe concentration was 1 μM; the nucleoprotein protein was 10 μg. 0.1% DMSO was used as the DMSO vehicle control. Error bars indicate mean±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p<0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: ANXA1 mimetic peptide Ac2-26 was purchased from Tocris Bioscience Company (Bristol, UK) and dissolved in PBS to make a 10 ng/μL concentration.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Concentration Assay, Control

Journal: iScience

Article Title: A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair

doi: 10.1016/j.isci.2023.108565

Figure Lengend Snippet: Schematic diagrams to elucidate the molecular mechanism of different dosages of PGE2 regulate the differential expression of ANXA1

Article Snippet: ANXA1 mimetic peptide Ac2-26 was purchased from Tocris Bioscience Company (Bristol, UK) and dissolved in PBS to make a 10 ng/μL concentration.

Techniques: Quantitative Proteomics

Journal: iScience

Article Title: A moderate dosage of prostaglandin E2-mediated annexin A1 upregulation promotes alkali-burned corneal repair

doi: 10.1016/j.isci.2023.108565

Figure Lengend Snippet:

Article Snippet: ANXA1 mimetic peptide Ac2-26 was purchased from Tocris Bioscience Company (Bristol, UK) and dissolved in PBS to make a 10 ng/μL concentration.

Techniques: Virus, Recombinant, Saline, Lysis, Protein Extraction, Luciferase, Plasmid Preparation, Mutagenesis, Expressing, Software