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: 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

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: ( A ) Graph of the correlation between the level of glycated hemoglobin HbA1c and the concentration of the SR-AI scavenger receptor in the samples. ( B ) Graph of the correlation between the level of glycated hemoglobin HbA1c and fluorescence of soluble pentosidine. ( C ) Graph of the correlation between the concentration of HDL and AGEs content in samples digested with proteinase K (the competitive ELISA test). Graph of the correlation between the levels of GFR. ( D ) and creatinine. ( E ) and the concentration of the SR-AI receptor in the serum samples. ( F ) Graph of the correlation between the level of uric acid and the fluorescence value of protein-bound total AGEs in the serum samples.

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: Concentration Assay, Fluorescence, Competitive ELISA

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: 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: The dependence of basic information about the health of patients on the concentration and fluorescence of AGEs and the concentration of AGE receptors. The statistically significant values are shown in red. PL—significance level for Levene’s test; PV – significance level for tke student’s t-test from the analysis of equality of variance; PNV – significance level for the students’s t-test of inequality of variance.

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: Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Indirect ELISA, Competitive ELISA