Journal: Cell reports

Article Title: Dendritic distribution of autophagosomes underlies pathway-selective induction of LTD

doi: 10.1016/j.celrep.2023.112898

Figure Lengend Snippet:

Article Snippet: (2 S ,2’ R ,3’ R )-2-(2’,3’-Dicarboxycyclopropyl) glycine (DCG-IV) , Bio-techne , Cat#0975/10.

Techniques: Virus, Recombinant, Sequencing, Plasmid Preparation, Software

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: Fetal bovine serum, RPMI 1640, TRIzol reagent, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester (DAPT) (#D5942), N-Benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1) (#553030), phalloidin fluorescein isothiocyanate labeled (P5282) and glutaraldehyde solution (#G5882) were obtained from Sigma-Aldrich (St Louis, Missouri).

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: Fetal bovine serum, RPMI 1640, TRIzol reagent, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester (DAPT) (#D5942), N-Benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1) (#553030), phalloidin fluorescein isothiocyanate labeled (P5282) and glutaraldehyde solution (#G5882) were obtained from Sigma-Aldrich (St Louis, Missouri).

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: AGE-induced Notch activation leads to fibrosis and podocyte foot process effacement. (A) Representative images of PAS and MT staining in mice glomeruli from with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1 treatment. Magnification ×400. Scale bar=50 µm. (B) The glomerular damage score was quantified as described in the ‘Research design and methods’ section. (C–F) Immunohistochemical staining for Col IV, α-SMA, and fibronectin in glomerular sections from mice treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Magnification ×400. Scale bar=50 µm. The intensity of glomerular expression of Col IV, α-SMA, and fibronectin was quantified using ImageJ (NIH). ****P<0.0001. (G) Representative TEM images of podocytes from mice treated with or without (CTL), AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Scale bar=1 µm. (H) Representative images of immunohistochemical staining for WT1 (podocytes) in the glomerulus from mice treated with or without (CTL) AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Magnification ×630. The arrows indicate detached podocytes. Scale bar=20 µm. (I) The percentage of glomerulus with detached podocytes was quantified with the help of ImageJ (NIH). ****P<0.0001. Data are presented as mean±SD. AGEs, advanced glycation end-products; Col IV, collagen IV; CTL, Control; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; MT, Masson’s trichrome; NIH, National Institutes of Health; PAS, periodic acid-Schiff; α-SMA, alpha-smooth muscle actin; TEM, transmission of electron microscopy.

Article Snippet: Fetal bovine serum, RPMI 1640, TRIzol reagent, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester (DAPT) (#D5942), N-Benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1) (#553030), phalloidin fluorescein isothiocyanate labeled (P5282) and glutaraldehyde solution (#G5882) were obtained from Sigma-Aldrich (St Louis, Missouri).

Techniques: Activation Assay, Staining, Immunohistochemical staining, Expressing, Transmission Assay, Electron Microscopy

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: Fetal bovine serum, RPMI 1640, TRIzol reagent, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester (DAPT) (#D5942), N-Benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1) (#553030), phalloidin fluorescein isothiocyanate labeled (P5282) and glutaraldehyde solution (#G5882) were obtained from Sigma-Aldrich (St Louis, Missouri).

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