anxa1 peptide ac2–26  (GenScript corporation)

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

Journal: Cell & Bioscience

Article Title: The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

doi: 10.1186/s13578-021-00679-1

Figure Lengend Snippet: Generating of AnxA1 −/− mice and detecting nociceptive sensation. a Gene targeting strategy. Two loxP sites were inserted on both sides of exon 6 of the AnxA1 locus followed by a FRT-flanked NEO cassette (F and NEO). To selectively delete AnxA1 gene in DRG neurons, we crossed AnxA1 loxP/ + mice with Avil cre mice to eventually obtain Avil cre - AnxA1 loxP/loxP ( AnxA1 −/− ) mice and littermate controls ( Avil cre - AnxA1 +/+ , Con.). b DNA band image of AnxA1 in control and AnxA1 −/− mice from PCR genotyping results. c The gross physical appearance and the body weight gain in control and AnxA1 −/− mice. d Immunofluorescent staining of DRG frozen sections isolated from control and AnxA1 −/− mice stained with antibody to ANXA1. Scale bar, 100 µm. Measurement of thermal ( e – g ) and mechanical ( h and i ) nociception between control and AnxA1 −/− mice. e Quantification of the thermal latency to radiant heat. f Quantification of the thermal latency to hot plate at 50 ℃ ( AnxA1 −/− versus control group, ***P < 0.001, Student’s t-test). g Quantification of the response latency to cold plate at 4 ℃. h Quantification of the threshold to von Frey filaments. i Quantitative analysis of mechanical pressure force in Randall-Selitto test ( AnxA1 −/− versus control group, P > 0.05, Student’s t-test). Measurement of chemical nociception (j and k) between control and AnxA1 −/− mice. j Quantitative analysis of the licking or biting duration over 10 min after unilateral injection of 40 μg menthol, 5% mustard oil or 25 μg capsaicin into the hindpaw of mice ( AnxA1 −/− versus control, **P < 0.01, Student’s t test). k Quantitative analysis of the licking or biting duration over 60 min after injection of 1% formalin into the hindpaw of mice ( AnxA1 −/− versus control, *P < 0.05, **P < 0.01, Student’s t-test). Quantitative analysis of the withdrawal latency to radiant heat in Hargreaves test ( l ) and threshold in von Frey test ( m ) after unilateral injection of CFA into the hindpaw of mice ( AnxA1 −/− versus control, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 10 in control group, n = 8 in AnxA1 −/− group). All data are represented as mean ± SD

Article Snippet: The ANXA1 mimetic peptide and the scrambled control peptide ANXA1 2–26 (Ac2-26, acetyl-AMVSEFLKQAWFIENEEQEYVQTVK) and the scramble peptide (acetyl-YESQFKAVWVE-INTQQMLKFEAEEV) were generated from GenicBio Limited (Shanghai, China) by using solid-phase stepwise synthesis.

Techniques: Control, Staining, Isolation, Randall–Selitto Test, Injection

Journal: Cell & Bioscience

Article Title: The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

doi: 10.1186/s13578-021-00679-1

Figure Lengend Snippet: AnxA1 deletion does not alter the expression of FPR2, TRPV1 and other nociceptors. a Representative images of double immunofluorescent staining and b Venn diagram of the percentages of FPR2 positive and TRPV1 positive neurons in DRG sections from AnxA1 −/− mice and WT littermates labeled for FPR2 and TRPV1 as indicated. Scale bar, 100 μm. c Representative western blots band images and the quantification of the indicated proteins d ANXA1 ( AnxA1 −/− versus control group, P = 0.45, Student’s t-test. n = 6 in control group, n = 6 in AnxA1 −/− group), and e TRPV1 AnxA1 −/− versus control group, P = 0.32, Student’s t-test. n = 6 in WT group, n = 6 in AnxA1 −/− group) in DRG tissues between AnxA1 −/− mice and control littermates. f Representative band images and the quantification of other nociceptors such as g TRPA1 and h TRPM8 in L4-6 DRG tissues between AnxA1 −/− mice and control littermates ( AnxA1 −/− versus control, P = 0.82, P = 0.85, Student’s t-test. n = 6 in control group, n = 6 in AnxA1 −/− group). The relative density of the protein band image in control group was normalized to 1. All data are represented as mean ± SD

Article Snippet: The ANXA1 mimetic peptide and the scrambled control peptide ANXA1 2–26 (Ac2-26, acetyl-AMVSEFLKQAWFIENEEQEYVQTVK) and the scramble peptide (acetyl-YESQFKAVWVE-INTQQMLKFEAEEV) were generated from GenicBio Limited (Shanghai, China) by using solid-phase stepwise synthesis.

Techniques: Expressing, Staining, Labeling, Western Blot, Control

Journal: Cell & Bioscience

Article Title: The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

doi: 10.1186/s13578-021-00679-1

Figure Lengend Snippet: AnxA1 deletion potentiates capsaicin induced calcium responses in DRG neurons. a Representative images of the calcium-dependent fluorescence before and after application with capsaicin (1 µM and 10 µM) in cultured DRG neurons between control mice (upper panels) and AnxA1 −/− mice (lower panels). Green color indicates basal intracellular Ca 2+ concentration (i.e. before depolarization) in Fura-2 dye loaded DRG neurons. Warmer colors (yellow, orange and red) indicate that the cytoplasmic Ca 2+ concentration is relatively higher. Scale bar, 100 μm. Representative traces showing the time course of changes in ratio (340/380) of calcium-dependent fluorescence during application of b and c capsaicin (1 µM and 10 µM), d and e mustard oil (10 µM and 100 µM), and f and g menthol (10 µM and 100 µM). Quantified data showing the percentage of calcium responses after application of c capsaicin (1 µM and 10 µM, AnxA1 −/− versus control group, ***P < 0.001, Student’s t-test. n = 9 in control group, n = 9 in AnxA1 −/− group), e mustard oil (10 µM and 100 µM, AnxA1 −/− versus control group, P = 0.99, P = 0.64, Student’s t-test. n = 8 in control group, n = 8 in AnxA1 −/− group), and g menthol (10 µM and 100 µM, AnxA1 −/− versus control group, P = 0.78, P = 0.78, Student’s t-test. n = 8 in control group, n = 9 in AnxA1 −/− group) in DRG neurons between AnxA1 −/− mice and control littermates. Responses percentage in control group was normalized to 10%. All data are represented as mean ± SD

Article Snippet: The ANXA1 mimetic peptide and the scrambled control peptide ANXA1 2–26 (Ac2-26, acetyl-AMVSEFLKQAWFIENEEQEYVQTVK) and the scramble peptide (acetyl-YESQFKAVWVE-INTQQMLKFEAEEV) were generated from GenicBio Limited (Shanghai, China) by using solid-phase stepwise synthesis.

Techniques: Fluorescence, Cell Culture, Control, Concentration Assay

Journal: Cell & Bioscience

Article Title: The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

doi: 10.1186/s13578-021-00679-1

Figure Lengend Snippet: AnxA1 deletion sensitizes capsaicin induced TRPV1 responses. Representative traces show the injected currents induced repetitive action potentials in control ( a ) and AnxA1 −/− ( b ) DRG neurons. c Quantification of action potential frequencies in control and AnxA1 -deficient DRG neurons after different currents injection (20, 100, and 200 pA). ( AnxA1 −/− versus control group, **** P < 0.0001, Two-way ANOVA repeated measures with Tukey’s multiple comparisons test. Control group, n = 10; AnxA1 −/− group, n = 8). Representative traces of d 10 μM capsaicin-gated currents, g 100 μM mustard oil-gated currents, j 100 μM menthol-gated currents and h 10 μM ATP-gated currents at -70 mV in control (black trace) and AnxA1 −/− (red trace) DRG neurons. Quantification of average current density after capsaicin e ( AnxA1 −/− versus control group, *** P < 0.001, Student’s t-test, n = 8 neurons in WT group and n = 8 neurons in AnxA1 −/− group from 3 cultures, respectively), mustard oil ( h ) ( AnxA1 −/− versus control group, P = 0.61, Student’s t-test, n = 10 neurons from 3 cultures in each group), and menthol ( k ) ( AnxA1 −/− versus control group, P = 0.94, Student’s t-test, n = 9 neurons from 3 cultures in each group), application measured at the current peak between control and AnxA1 −/− group. Time constants Tau of capsaicin ( f ), mustard oil ( i ), and menthol ( l )-induced activation and deactivation at -70 mV in control and AnxA1 -deficient DRG neurons ( AnxA1 −/− versus control group, capsaicin: P = 0.81, P = 0.55; mustard oil: P = 0.79, P = 0.87; menthol: P = 0.99, P = 0.59, Student’s t-test). All data are represented as mean ± SD

Article Snippet: The ANXA1 mimetic peptide and the scrambled control peptide ANXA1 2–26 (Ac2-26, acetyl-AMVSEFLKQAWFIENEEQEYVQTVK) and the scramble peptide (acetyl-YESQFKAVWVE-INTQQMLKFEAEEV) were generated from GenicBio Limited (Shanghai, China) by using solid-phase stepwise synthesis.

Techniques: Injection, Control, Activation Assay

Journal: Cell & Bioscience

Article Title: The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

doi: 10.1186/s13578-021-00679-1

Figure Lengend Snippet: ANXA1 mimics Ac2-26 increases intracellular Ca 2+ , and activates PLCβ via FPR2. a Representative images of the calcium-dependent fluorescence 30, 60 and 120 s after application with scramble (3.3 μM), Ac2-26 (3.3 μM) and Boc2 (10 μM) + Ac2-26 (3.3 μM) in cultured AnxA1 −/− DRG neurons. Green color indicates basal intracellular Ca 2+ concentration (i.e. before depolarization) in Fura-2 dye loaded DRG neurons. Warmer colors (yellow, orange and red) indicate that the cytoplasmic Ca 2+ concentration is relatively higher. Scale bar, 100 μm. b Representative traces showing the time course of changes in ratio (340/380) of calcium-dependent fluorescence during application of scramble (3.3 μM), Ac2-26 (3.3 μM) and Boc2 (10 μM) + Ac2-26 (3.3 μM). c , Quantification of the intracellular Ca 2+ concentration ([Ca 2+ ]) responding to scramble (3.3 μM), Ac2-26 (3.3 μM) and Boc2 (10 μM) + Ac2-26 (3.3 μM) (Ac2-26 versus scramble group, *** P < 0.001; Ac2-26 versus Boc2 + Ac2-26 group, **P < 0.01, one-way ANOVA, post hoc Student’s t test. n = 8 in scramble group, n = 9 in Ac2-26 group and n = 8 in Boc2 + Ac2-26 group). d Representative protein band images of the pPLCβ and PLCβ after application with scramble (3.3 μM), Ac2-26 (3.3 μM) and Boc2 (10 μM) + Ac2-26 (3.3 μM) in cultured AnxA1 −/− DRG neurons. β-actin was used as the internal reference protein. e Quantification of the relative protein intensities of pPLCβ among scramble, Ac2-26 and Boc2 + Ac2-26 groups. The data of relative intensities in scramble group were normalized to 1 (Ac2-26 versus scramble group, *** P < 0.001; Ac2-26 versus Boc2 + Ac2-26 group, *** P < 0.001, one-way ANOVA, post hoc Tukey’s multiple comparisons test, n = 5 in each group). All data are represented as means ± SD

Article Snippet: The ANXA1 mimetic peptide and the scrambled control peptide ANXA1 2–26 (Ac2-26, acetyl-AMVSEFLKQAWFIENEEQEYVQTVK) and the scramble peptide (acetyl-YESQFKAVWVE-INTQQMLKFEAEEV) were generated from GenicBio Limited (Shanghai, China) by using solid-phase stepwise synthesis.

Techniques: Fluorescence, Cell Culture, Concentration Assay

Journal: Cell & Bioscience

Article Title: The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

doi: 10.1186/s13578-021-00679-1

Figure Lengend Snippet: Ac2-26 enhances interaction between CaM and TRPV1 via FPR2. a Representative images of double immunofluorescent staining after application with scramble (3.3 μM), Ac2-26 (3.3 μM) and Boc2 (10 μM) + Ac2-26 (3.3 μM) in cultured AnxA1 −/− DRG neurons. The areas in the dotted box in merge pictures zoom in and present in magnify panels. Scale bar, 50 μm in TRPV1, CaM and merge pictures, 25 μm in magnify pictures. b Quantification of the fluorescent intensities of TRPV1 positive and CaM positive (TRPV1 + /CaM + ) cells among scramble, Ac2-26 and Boc2 + Ac2-26 groups. (Ac2-26 versus scramble group, ****P < 0.0001; Ac2-26 versus Boc2 + Ac2-26 group, ***P < 0.001, one-way ANOVA, post hoc Student’s t test. n = 10 in each group). All data are represented as mean ± SD. c and d Representative Western blots (WB) band images of immunoprecipitation (IP) experiments in cultured AnxA1 −/− DRG neurons treated with scramble (3.3 μM), Ac2-26 (3.3 μM) and Boc2 (10 μM) + Ac2-26 (3.3 μM), respectively. c TRPV1 antibody incubated immunoprecipitations were detected with CaM and TRPV1 antibodies by western blotting. d CaM antibody incubated immunoprecipitants were detected with TRPV1 and CaM antibodies by western blotting. Input, 10 μg protein of the extracts without IP was loaded. IgG, immunoprecipitants incubated with nonspecific IgG (IgG) as negative control. e Representative traces of capsaicin-evoked currents with co-application of scramble, Ac2-26 and Boc2 + Ac2-26 in cultured AnxA1 −/− DRG neurons. f Quantification of the fold change peak current in scramble, Ac2-26 and Boc2 + Ac2-26 groups. The data of fold change peak current in scramble group were normalized to 1 (Ac2-26 versus scramble group, ****P < 0.0001; Ac2-26 versus Boc2 + Ac2-26 group, ****P < 0.0001, one-way ANOVA, post hoc Tukey’s multiple comparisons test, n = 8 in each group)

Article Snippet: The ANXA1 mimetic peptide and the scrambled control peptide ANXA1 2–26 (Ac2-26, acetyl-AMVSEFLKQAWFIENEEQEYVQTVK) and the scramble peptide (acetyl-YESQFKAVWVE-INTQQMLKFEAEEV) were generated from GenicBio Limited (Shanghai, China) by using solid-phase stepwise synthesis.

Techniques: Staining, Cell Culture, Western Blot, Immunoprecipitation, Incubation, Negative Control