lentiviral expression vectors containing rage-specific shrnas and green fluorescent protein (gfp)  (Genechem)

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: Oncotarget

Article Title: Glucose-derived AGEs enhance human gastric cancer metastasis through RAGE/ERK/Sp1/MMP2 cascade

doi: 10.18632/oncotarget.22185

Figure Lengend Snippet: (A) mRNA expression profile of RAGE in different gastric cancer cells. (B) Protein expression and quantification of RAGE in gastric cancer cells. (C) RAGE was stably knocked down in SGC7901 cells. (D) The peritoneal nodules in nude mice administered with glucose-derived AGEs for two months. (E) The quantification of peritoneal nodules. ( ** P <0.01). (F) The inhibitory effect of RAGE knock-down on the AGEs-induced Sp1 and MMP2 expression in vivo .

Article Snippet: In order to stably knock-down RAGE expression of SGC7901 cells, lentiviral expression vectors containing RAGE-specific shRNAs and green fluorescent protein (GFP) were constructed by GeneChem (Shanghai, China).

Techniques: Expressing, Stable Transfection, Derivative Assay, Knockdown, In Vivo

Journal: Oncotarget

Article Title: Glucose-derived AGEs enhance human gastric cancer metastasis through RAGE/ERK/Sp1/MMP2 cascade

doi: 10.18632/oncotarget.22185

Figure Lengend Snippet: (A) Wound healing assays with SGC7901 treated with 100μg/ml AGEs or unmodified BSA was recorded at 0 and 48 hours. (B) The mean distances between wound edges of SGC7901 cells at 0 and 48 hours. (C, D) Transwell migration and invasion assays for SGC7901 cells treated with 100μg/ml AGEs or unmodified BSA for 2 days. (E, F) The number of migration and invasion cells. All experiments were carried out in triplicate. ( ** p <0.05).

Article Snippet: In order to stably knock-down RAGE expression of SGC7901 cells, lentiviral expression vectors containing RAGE-specific shRNAs and green fluorescent protein (GFP) were constructed by GeneChem (Shanghai, China).

Techniques: Migration

Journal: Oncotarget

Article Title: Glucose-derived AGEs enhance human gastric cancer metastasis through RAGE/ERK/Sp1/MMP2 cascade

doi: 10.18632/oncotarget.22185

Figure Lengend Snippet: (A) Representative images of immunohistochemistry showed overexpression of Sp1 in human gastric cancer tissues (200×). (B) Suppression of Sp1 protein expression in SGC7901 cells with Sp1sepcific siRNAs. (C, D) The inhibitory effects of Sp1 knockdown on AGEs-induced cells migration and invasion. (E, F) Reduction of AGEs-induced MMP2 protein expression and enzymatic activity in SGC7901 cells by down-regulating Sp1 expression. All operations were repeated three times. ( ** p <0.05).

Article Snippet: In order to stably knock-down RAGE expression of SGC7901 cells, lentiviral expression vectors containing RAGE-specific shRNAs and green fluorescent protein (GFP) were constructed by GeneChem (Shanghai, China).

Techniques: Immunohistochemistry, Over Expression, Expressing, Knockdown, Migration, Activity Assay