Journal: Experimental cell research

Article Title: Regulation of the AGEs-induced inflammatory response in human periodontal ligament cells via the AMPK/NF-κB/ NLRP3 signaling pathway.

doi: 10.1016/j.yexcr.2024.113999

Figure Lengend Snippet: Fig. 3. Indispensability of RAGE in Inflammasome Activation. (A) Transcript expression of RAGE in control HPDLCs versus HPDLCs stimulated with AGEs for 24 h. (B) Protein expression (left) and quantitative analysis (right) of RAGE in control group compared to HPDLCs post 6, 8, and 24 h of culture with AGEs. (C) and (D) A noteworthy diminution in both gene and protein expression of RAGE was observed in RAGE siRNA group as opposed to control group or control siRNA group. (E) Western Blot outcomes and quantitation of NLRP1, NLRP3, and ASC post 48 h of AGEs stimulation with or without RAGE silencing. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the pursuit of targeted gene silencing, human periodontal ligament cells (HPDLCs) were transfected with RAGE-specific siRNA (Santa Cruz Biotechnology, sc-156124, Dallas, TX, USA) at a concentration of 116.7 nmol/L.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitation Assay

Journal: BMJ Open Diabetes Research & Care

Article Title: Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria

doi: 10.1136/bmjdrc-2020-001203

Figure Lengend Snippet: AGEs induce Notch signaling in podocytes. (A) Non-tryptophan AGE fluorescence was measured to demonstrate the formation of AGEs in D-glucose+BSA preparations at Ex: 370 nm and Em: 400–500 nm. (B) Quantification of free amines in BSA and D-glucose+BSA preparations of various concentrations (50–200 μg/mL of BSA) by TNBS assay. (C) BSA and D-glucose+BSA preparations were subjected to SDS-PAGE and stained with Coomassie blue. The arrowhead indicates high-molecular-weight aggregates in the stacking region of the gel. (D) Immunoblots showing the presence of AGEs in D-glucose+BSA preparations. M indicates the standard protein marker (D, E). (E) Immunoblots showing the expression of RAGE, NICD1, and β-actin in HPC cells treated with AGEs (25–200 µg/mL) for 48 hours. (F) Immunoblots showing the expression of RAGE, NICD1, and β-actin in HPC treated with BSA alone (25–200 µg/mL) for 48 hours. (G) Immunoblots showing the expression of RAGE, JAG1, NICD1, HES1, and β-actin in HPC treated with AGEs (100 µg/mL) for indicated time intervals (24–72 hours). (E–G) The fold expression was presented after normalizing with β-actin. (H) γ-secretase activity in HPC treated with (50–200 µg/mL) or without AGEs for 48 hours (n=6). *P<0.05, ****P<0.0001. Data are presented as mean±SD (n=3). AGEs, advanced glycation end-products; a.u, arbitrary unit; BSA, bovine serum albumin; CTL, control; Em, emission; Ex, excitation; HES1, hairy and enhancer of split homolog1; HPC, human podocyte; JAG1, jagged1; NICD1, Notch intracellular domain; RAGE, receptor for AGEs; SDS-PAGE, sodium dodecyl sulfate poly-acrylamide gel electrophoresis; TNBS, trinitrobenzene sulfonic acid.

Article Snippet: Scrambled RNA and small interfering RNA (siRNA) specific to receptor for AGEs (RAGE) and Notch1 were purchased from Kaneka Eurogentec (Belgium).

Techniques: Fluorescence, SDS Page, Staining, Molecular Weight, Western Blot, Marker, Expressing, Activity Assay

Journal: BMJ Open Diabetes Research & Care

Article Title: Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria

doi: 10.1136/bmjdrc-2020-001203

Figure Lengend Snippet: AGE-activated Notch signaling promotes EMT in podocytes. (A–C) qRT-PCR analysis showing the expression of (A) NOTCH1, (B) JAG1, and (C) HES1 in HPC treated with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1. β-actin was used as an internal control. **P<0.01, ***P<0.0001. (D) Immunoblots showing the expression of NICD1, JAG1, HES1, and β-actin in HPC treated with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1 (48 hours). The fold change values were presented after normalizing with β-actin. (E) HPC cells transfected with specific siRNA targeting RAGE and Notch1 or scramble RNA (Scr) were subjected to immunoblotting for RAGE, NOTCH, JAG1, and NICD1. (F) Expression of E-CAD, N-CAD, and vimentin in HPC (CTL, AGEs, AGEs+DAPT, and AGEs+FPS-ZM1) was analyzed by qRT-PCR. The expression of β-actin was used as an internal control. ****P<0.0001. (G) Immunoblotting analysis of expression of E-CAD, N-CAD and vimentin in HPC (CTL, AGEs, AGEs+DAPT, and AGEs+FPS-ZM1). (E, G) The fold change values were presented after normalizing with β-actin expression. (H) Phalloidin staining of podocytes showing F-actin arrangement. The white arrows indicate filopodia formation. Scale bar=20 µm. (I) Quantification of the average number of filopodia formation observed from the phalloidin staining (n=16). ****P<0.0001. Data are presented as mean±SD (n=3). AGEs, advanced glycation end-products; CTL, control; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; E-CAD, E-Cadherin; EMT, epithelial to mesenchymal transition; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; HES1, hairy and enhancer of split gene1; HPC, human podocyte; JAG1, jagged1; N-CAD, N-Cadherin; NICD1, Notch intracellular domain; qRT-PCR, quantitative reverse transcription-PCR; RAGE, receptor for AGEs; siRNA, small interfering RNA.

Article Snippet: Scrambled RNA and small interfering RNA (siRNA) specific to receptor for AGEs (RAGE) and Notch1 were purchased from Kaneka Eurogentec (Belgium).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Staining, Small Interfering RNA

Journal: BMJ Open Diabetes Research & Care

Article Title: Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria

doi: 10.1136/bmjdrc-2020-001203

Figure Lengend Snippet: AGEs induce Notch signaling in mice glomeruli and inhibition of γ-secretase and RAGE ameliorates EMT in mice kidney. (A) Immunofluorescence for the nuclear co-localization study of NICD1 (Cy3, red) and HES1 (Cy5, far-red) in HPC treated with or without (CTL) AGEs, AGEs+DAPT and AGEs+FPS-ZM1. Magnification ×630. Scale bar=20 µm. (B) Immunoblots for NICD1, HES1 and histone H2B, the nuclear extract of HPC, treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1. The fold change values were presented with the expression of the respective genes after normalizing with H2B. (C) Immunostaining for RAGE expression in mice glomeruli from with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6, each group). Magnification ×630. Scale bar=20 µm. (D) Double immunostaining with anti-NICD1 (Alexa Fluor 555) and anti-WT1 (Cy3, red) in glomerular sections from with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 treatment (n=6, each group). Magnification ×630. Scale bar=20 µm. (E) Immunoblotting analysis for NICD1, JAG1, HES1, and β-actin in mice glomerular lysates from with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 treated mice (n=6, each group). The fold change values were presented with the expression of the respective genes after normalizing with β-actin. (F) Immunoblotting analysis for E-CAD, N-CAD, vimentin, and β-actin in glomerular lysates from with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1 treated mice. The fold change values were presented with the expression of the respective genes after normalizing with β-actin. Data are presented as mean±SD (n=3). AGEs, advanced glycation end-products; CTL, control; DAPI, 4′,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; E-CAD, E-Cadherin; EMT, epithelial to mesenchymal transition; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; HES1, hairy and enhancer of split gene1; HPC, human podocyte; JAG1, jagged1; N-CAD, N-Cadherin; NICD1, Notch intracellular domain; RAGE, receptor for AGEs.

Article Snippet: Scrambled RNA and small interfering RNA (siRNA) specific to receptor for AGEs (RAGE) and Notch1 were purchased from Kaneka Eurogentec (Belgium).

Techniques: Inhibition, Immunofluorescence, Western Blot, Expressing, Immunostaining, Double Immunostaining

Journal: BMJ Open Diabetes Research & Care

Article Title: Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria

doi: 10.1136/bmjdrc-2020-001203

Figure Lengend Snippet: Blockade of Notch and RAGE protects mice from proteinuria, and elevated levels of AGEs correlate with Notch activation in people with DN. (A) AGEs alter podocyte permeability in vitro. Albumin permeability across the podocyte monolayer was determined after 48 hours of exposure to AGEs (n=3). ****P<0.0001. (B) Immunoblotting study for podocin and nephrin expression in glomerular lysates and (C) by immunohistochemical staining for podocin in the glomerulus from mice treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Magnification ×630. Scale bar=20 µm. The fold change values were presented with the expression of the respective genes after normalizing with β-actin. (D) UACR was estimated in mice treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). ****P<0.0001. (E) Immunoblotting analysis for AGEs in urine samples of DN (n=5) and non-diabetic group (n=3). The arrowhead indicates the positive staining for AGEs in the urine samples of a patient with DN. (F) Immunohistochemical staining of glomerular serial sections from patients with DN (n=16) and non-diabetic groups (n=10) for AGEs (DyLight488, green), RAGE (Cy3, red), NICD1 (Alexa Fluor 555), and HES1 (Cy5, far-red). Magnification ×630. Scale bar=20 µm. (G) A proposed model depicting the adverse effect of AGEs on podocytes. Activation of Notch signaling via AGE–RAGE interaction induces the thickening of GBM, EMT, and dehiscence of podocytes, which eventually result in impaired glomerular permselectivity and proteinuria. AGEs, advanced glycation end-products; CTL, control; DAPI, 4′,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; DN, diabetic nephropathy; EMT, epithelial to mesenchymal transition; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; GBM, glomerular basement membrane; HES1, hairy and enhancer of split gene1; NICD1, Notch intracellular domain; RAGE, receptor for AGEs; SD, slit-diaphragm; UACR, urinary albumin to creatinine ratio.

Article Snippet: Scrambled RNA and small interfering RNA (siRNA) specific to receptor for AGEs (RAGE) and Notch1 were purchased from Kaneka Eurogentec (Belgium).

Techniques: Activation Assay, Permeability, In Vitro, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: RAGE Up-Regulation Differently Affects Cell Proliferation and Migration in Pancreatic Cancer Cells

doi: 10.3390/ijms21207723

Figure Lengend Snippet: ( A , B ) RAGE expressing FLR2 and FLR3 Panc-1 cells have higher proliferation rates than WT Panc-1 cells. ( A ) Cell proliferation was assessed by Trypan blue exclusion assay. Black bars: number of cells at the time of seeding; gray bars: number of cells after a 48 h incubation. ( B ) Cell proliferation was assessed using resazurin fluorescence 48 h after cell seeding. ( C ) RAGE silencing reduces RAGE expression levels in FLR2 Panc-1 cells. FLR2 Panc-1 cells were transfected using RAGE specific siRNAs or control (Ctrl) siRNAs. ( D ) RAGE silencing reduces the proliferation of FLR2 Panc-1 cells. FLR2 Panc-1 cells were silenced with either RAGE specific siRNAs (RAGE siRNA) and scrambled siRNAs (Ctrl siRNAs). ( E ) RAGE inhibition reduces the proliferation of FLR2 Panc-1 cells. Cell proliferation was assessed using resazurin fluorescence. Cells were incubated with media only (Ctrl), 0.2% DMSO, 1 µM or 10 µM FPS-ZM1. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Transfection was performed using either RAGE specific siRNAs (sc-36374) or a scrambled control siRNA (sc-37007, Santa Cruz Biotechnologies, Dallas, TX, USA) using the instructions of the manufacturer.

Techniques: Expressing, Trypan Blue Exclusion Assay, Incubation, Fluorescence, Transfection, Control, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: RAGE Up-Regulation Differently Affects Cell Proliferation and Migration in Pancreatic Cancer Cells

doi: 10.3390/ijms21207723

Figure Lengend Snippet: ( A , B ) RAGE expression decreases cell migration in FLR2 Panc-1 cells. Cell migration was assessed using the Boyden chamber migration assay. The percentage of migrated cells 24 h after seeding was estimated using resazurin. ( B ) RAGE expression reduces wound healing in FLR2 Panc-1 cells assay with WT and FLR2 Panc-1 cells. Cells were images at 0 h, 24 h, and 48 h following the formation of the wound. Representative images are shown. ( C ) RAGE silencing in FLR2 Panc-1 cells reverses wound healing inhibition in FLR2 Panc-1 cells. Wound healing assay with FLR2 Panc-1 cells that had been either transfected with RAGE specific siRNAs or scrambled siRNAs, or treated with either vehicle (0.2% DMSO) or 10 µM FPS-ZM1. Images were taken at t = 0 h and t = 48 h. Representative images are shown. *** p < 0.001.

Article Snippet: Transfection was performed using either RAGE specific siRNAs (sc-36374) or a scrambled control siRNA (sc-37007, Santa Cruz Biotechnologies, Dallas, TX, USA) using the instructions of the manufacturer.

Techniques: Expressing, Migration, Inhibition, Wound Healing Assay, Transfection