Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Involvement of the coupled RAGE ligand/receptor in human corneal epithelial (HCE) cell wound healing. Representative images of scratch assays performed on HCE cells (left panel) treated with HMGB1 (high mobility group box 1, 100 ng/mL) ( A ) or AGEs (advanced glycation end products) (10/100/200 µg/mL) (B , C ). Percentage of residual wound area (right panel) after treatment with HMGB1 (100 ng/mL) ( A ) or AGEs (10/100/200 µg/mL) ( B , C ), compared with 0 hours and standardized to the untreated condition (100%) (n = 5 experiments, each conducted in duplicate). ( D ) Representative images of scratch assays performed on HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) or siRNA control (Scramble) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) (left panel). Percentage of the residual wound area of HCE cells transfected with siRNA against RAGE (100 nM) for 36 hours and then treated with AGEs (100 µg/mL), compared with 0 h (right panel) (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney test after a nonparametric ANOVA analysis; * P < 0.05; ** P < 0.01; *** P < 0.005; ns: not significant.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Transfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Functionality of the RAGE pathway. Characterization of the RAGE ( A ) mRNA and ( B ) protein expression in human cornea, primary human epithelial cells (mRNA only), and the HCE cell line (mRNA and protein) evaluated by ( A ) RT-PCR, ( B ) immunofluorescence, and ( B ) western blotting. For RT-PCR, negative controls (NC) were performed ( A ) without cDNA. ( B , left panel) Representative images of RAGE expression ( green ) in human corneas (top panel) and HCE cells (bottom panel). Nuclei were stained with Hoechst ( blue ); NC (left) were obtained by incubating HCE cells without primary antibody. ( B , right panel) Western blot experiments identified the RAGE protein at the described molecular weight (46 kDa). ( C ) Functionality of the NF-κB pathway (by luciferase reporter gene activity) was measured after treatment of HCE cells with AGEs (100 µg/mL) for 45 minutes (n = 5 experiments, each conducted in duplicate) (right panel). Positive controls (T+) were obtained by co-transfection with pMEKK (n = 5 experiments, each conducted in duplicate) (left panel). Each bar graph shows mean ± SEM. Mann-Whitney; * P < 0.05.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Staining, Molecular Weight, Luciferase, Activity Assay, Cotransfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Cx43 expression is induced by the AGEs/RAGE axis in HCE cells. Characterization of Cx43 protein expression ( arrow ) by immunostaining (bottom panel). Cells incubated without primary antibody served as a negative control (NC). ( B ) Relative quantification of Cx43 mRNA expression in untreated HCE cells 0, 6, and 24 hours after scratch wounding. Results were expressed as a ratio of the 0-hour condition (n = 5 experiments, each conducted in duplicate). ( C ) Quantification of Cx43 mRNA expression in HCE cells treated with AGEs (100 µg/mL) for 6 hours or 24 hours without scratch wounding (left panel) or with scratch wounding (right panel). Cells not treated with AGEs served as a control. Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate). (Left panel) Quantification of Cx43 mRNA expression in HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) for 6 hours. Results were expressed as a ratio of the scrambled siRNA condition (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney after a nonparametric ANOVA analysis; * P < 0.05; ns: not significant. ( D ) Relative quantification of Cx43 protein expression in HCE cells following treatment with AGEs (100 µg/mL) after scratch wounding. Untreated cells served as a control. Results were expressed as a ratio of the 0-hour, unwounded condition (n = 5 experiments) (top panel). Quantification of Cx43 protein expression in HCE cells treated with AGEs (100 µg/mL) for 12 and 24 hours after scratch wounding (bottom panel). Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate) (** P < 0.01). (E) Characterization by immunostaining of Cx43 protein expression according to the distance from the wound, time, and treatment with AGEs (100 µg/mL). Staining of Cx43 protein expression in HCE cells treated near the wound margins (left panel) and behind the wound (right panel). Cells not treated with AGEs (100 µg/mL) served as a control. Cells incubated without primary antibody served as a negative control (NC).

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Immunostaining, Incubation, Negative Control, Transfection, MANN-WHITNEY, Staining

Journal: Scientific Reports

Article Title: A novel modified-curcumin 2.24 resolves inflammation by promoting M2 macrophage polarization

doi: 10.1038/s41598-023-42848-x

Figure Lengend Snippet: Effects of CMC2.24 on Resolvin D 1 and soluble RAGE. The effects of CMC2.24 on the concentrations (pg/mL) of RvD 1 ( a ) and sRAGE ( b ) in rat peritoneal Mφs challenged by LPS-alone, AGE-alone, or the “two-hit”, respectively; and treated with different concentrations (2 or 5 μM) of CMC2.24 at 18-h. N, the normal group. L, the LPS-alone challenged group. L + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. A, the AGE-alone challenged group. A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. L + A, the “two-hit” challenged group. L + A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. Each value represents the mean (n = 3/group) ± the standard error. *p < 0.05, **p < 0.005, ***p < 0.001, values were compared between N and L/A/(L + A)-challenged at 18-h. # p < 0.05, ### p < 0.001, values were compared between L/A/(L + A)-challenged and L/A/(L + A) + 2.24 (2 or 5)-treated groups at 18-h.

Article Snippet: RvE 1 (Cat# MBS2604861) and soluble receptor of AGE (sRAGE) (Cat# MBS722302) were purchased from MyBioSource, Inc. (San Diego, CA, USA), and LxA 4 (Cat# 407,010) was purchased from Neogen Corporation (Lexington, Kentucky, USA).

Techniques:

Journal: Scientific Reports

Article Title: Advanced glycation end products and their receptors in serum of patients with type 2 diabetes

doi: 10.1038/s41598-021-92630-0

Figure Lengend Snippet: Graphs of the dependence of AGEs fluorescence or content of sRAGE in the samples and occurrence diabetic complications in patients. The numbers given are the mean value of the results and the standard error of the mean (Mean ± SE). ( A ) The dependence of total AGEs fluorescence on the type of diabetes; (number of subjects = 49; Type 1 (1,167,393 ± 70,632 a.u.), Type 2 (1,517,352 ± 98,785 a.u.)). ( B ) The dependence of total soluble AGEs fluorescence on the occurrence of atherosclerosis; (n = 47; YES (1,581,783 ± 145,154 a.u.), NO (1,179,192 ± 107,389 a.u.)). ( C ) The dependence of total AGEs fluorescence on the occurrence of macroangiopathy; (n = 58; YES (1,511,867 ± 619,228 a.u.), NO (1,105,011 ± 228,314 a.u.)). ( D ) The dependence of total soluble AGEs fluorescence on the occurrence of microangiopathy; (n = 47; YES (1,764,035 ± 207,484 a.u.), NO (1,205,829 ± 68,639 a.u.)). ( E ) The dependence of total soluble AGEs fluorescence on the occurrence of retinopathy; (n = 47; YES (2,749,771 ± 757,703 a.u.), NO (1,327,710 ± 66,108 a.u.)). ( F ) The dependence of soluble pentosidine fluorescence on the occurrence of retinopathy; (n = 47; YES (3,320,508 ± 527,192 a.u.), NO (3,069,839 ± 117,568 a.u.)). ( G ) The dependence of AGEs concentration determined in the competition ELISA test on the occurrence of ischemic stroke; (n = 39; YES (0.9252 ± 0.0687 mg/ml), NO (0.5244 ± 0.0517 mg/ml)). ( H ) The dependence of the concentration of sRAGE receptors on the occurrence of ischemic heart disease; (n = 49; YES (15,467 ± 3967 pg/ml), NO (7404 ± 1628 pg/ml)).

Article Snippet: sRAGE was determined using the ELISA kit for the sRAGE human receptor (MyBioSource, cat. no. MBS766075) according to the manufacturer's instructions.

Techniques: Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: An Immunogenic Peptide in the A-box of HMGB1 Protein Reverses Apoptosis-induced Tolerance through RAGE Receptor * ^♦

doi: 10.1074/jbc.M113.541474

Figure Lengend Snippet: A defined region in HMGB1 A-box binds to RAGE extracellular domains to reverse the tolerogenic program of DCs fed apoptotic cells. A, HMGB1 dependent reversal of tolerance does not require TLRs-3, -4, -6, and -9. The assay was carried out as in Fig. 2B using DCs from WT, Tlr3−/−, Tlr4−/−, Tlr6−/−, or Tlr9−/− mice. B, the HMGB1 A-box signals on DCs through RAGE to reverse tolerance. The assay was conducted as in Fig. 2B using DCs from Rage+/+ or Rage−/− mice. C, RAGE is composed of a short cytosolic tail (CT) involved in signal transduction, a transmembrane domain (TM), which anchors the protein to the cell membrane, two constant domains (C1 and C2), and a variable domain (V). sRAGE lacks the CT and TM domains. D, top, caspase-1-generated HMGB1 A-box fragment amino acid sequence and overlapping synthetic peptides used to map binding sites to RAGE. Bottom, representative SPR for recombinant A-box fragment (0–100 nm; 2-fold dilution series) and A-box synthetic peptides (0–10 μm; 2-fold dilution series) titrated over amine-coupled RAGE surfaces (∼250 RU each) in HBS-EP at 50 μl/min (1-min association + 3-min dissociation). Apparent equilibrium dissociation constants (KD) are indicated. N/A = no significant binding. E, A-box activity on reversal of tolerance correlates with a RAGE-binding region between amino acids 23–50. Overlapping synthetic A-box peptides (1 μg/ml) were assayed for activity in reversing tolerance as in Fig. 2B; recombinant HMGB1 was used as a positive control.

Article Snippet: Recombinant mammalian sRAGE was from Prospec (East Brunswick, NJ), and V-C1 and V-C1-C2 constructs were generated by Dr. Tsian Xiao at the National Institutes of Health (Bethesda, MD).

Techniques: Transduction, Generated, Sequencing, Binding Assay, Recombinant, Activity Assay, Positive Control

Journal: International Journal of Stem Cells

Article Title: CRISPR/Cas9 Edited sRAGE-MSCs Protect Neuronal Death in Parkinson’s Disease Model

doi: 10.15283/ijsc18110

Figure Lengend Snippet: Generation and characterization of sRAGE secreting UCB-MSC. (A) The illustration picture represents the gene information of pZDonor-AAVS1 puromycin vector. Each arrow describes certain gene. (B) The illustration of the sRAGE insertion coding sequence. (C) Genome integration was confirmed by Junction PCR with genomic DNAs of UCB-MSCs which were transfected with mock, GFP and sRAGE containing pZDonor-AAVS1 plasmids. (D) Immunoblot analysis of supernatant and extract from UCB-MSC cells transfected with mock (lane 1) and FLAG-tagged sRAGE in pZDonor-AAVS1 vector (lane 2). β -actin loaded as a positive control. The secretion of human sRAGE levels (E) was confirmed with ELISA. ***p<0.001.

Article Snippet: Human soluble receptor for advanced glycosylation end products (sRAGE) ELISA kit (Aviscera Bioscience) was used to quantify the amount of secreted sRAGE.

Techniques: Plasmid Preparation, Sequencing, Transfection, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay