ager  (OriGene)

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