Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: (a) Representative Western blot and (b) bar graph quantification of APOM in scrambled control (shSC) and APOM knockdown (shAPOM) human podocytes (n=3). Bar graph quantification of (c) apoptosis and (d) cytotoxic cell death in shAPOM podocytes (n=4–5). (e) Confocal images of lipid droplets and (f) bar graph quantification of lipid droplet number in shAPOM podocytes (n = 3). HCS CellMask (green), DAPI (blue), HCS neutral LipidTox (red); 20× magnification. (g) Representative Western blot and (h) bar graph quantification of S1PR4 overexpressing (S1PR4 OE) (n=3). Bar graph quantification of (i) apoptosis and (j) cytotoxic cell death in S1PR4 OE podocytes (n=4). (k) Confocal images of lipid droplets and (l) bar graph quantification of lipid droplet number in S1PR4 OE podocytes (n = 3). Nile Red (green), DAPI (blue), CellMask HCS and phalloidin (red); 20× magnification.

Article Snippet: The knockdown control podocyte cell line was generated by infection with scrambled shRNA (Origene, TR30021V).

Techniques: Knockdown, Over Expression, Western Blot, Control

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Human RAGE antibody protects against AGE-mediated podocyte dysfunction.

doi: 10.1093/ndt/gfs005

Figure Lengend Snippet: Figure 2 Inflammation. Immunofluorescence staining of podocytes to investigate the effect of ±hRAGEab after 48 h of incubation on inflammation in the control, PDF and GDP group. Upper panel shows bar graph analysis of NFκB-activated cells, lower panel shows representative images of the staining, scale bar represents 50 μm (A). IL- 6 ELISA was performed shown in (B). **P < 0.01 versus −hRAGEab, *P < 0.05 versus −hRAGEab.

Article Snippet: Afterwards, human podocytes were lysed, protein concentration was assessed by Pierce BCA Protein Assay Kit (Thermo Scientific, Langenselbold, Germany) and after being boiled in sodium dodecyl sulphate (SDS), sample buffer 5–20 μg of lysate was separated by 8–12% SDS– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane.

Techniques: Staining, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Human RAGE antibody protects against AGE-mediated podocyte dysfunction.

doi: 10.1093/ndt/gfs005

Figure Lengend Snippet: Figure 1 CML expression. Immunofluorescence staining of podocytes to investigate the effect of ±hRAGEab after 48 h of incubation on the expression of CML in the control, PDF and GDP group, scale bar represents 50 μm.

Article Snippet: Afterwards, human podocytes were lysed, protein concentration was assessed by Pierce BCA Protein Assay Kit (Thermo Scientific, Langenselbold, Germany) and after being boiled in sodium dodecyl sulphate (SDS), sample buffer 5–20 μg of lysate was separated by 8–12% SDS– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane.

Techniques: Expressing, Staining, Incubation, Control

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Human RAGE antibody protects against AGE-mediated podocyte dysfunction.

doi: 10.1093/ndt/gfs005

Figure Lengend Snippet: Figure 3 Cytoskeleton-associated proteins and integrin. Immunofluorescence staining of podocytes to investigate the effect of ±hRAGEab after 48 h of incubation on phalloidin (FITC) and synaptopodin (Cy3) in the control, PDF and GDP group, scale bar represents 20 μm (A), the quantitation of the organization of the actin cytoskeleton (B) and activated αvβ3 integrin (C). ***P < 0.001 versus −hRAGEab, **P < 0.01 versus −hRAGEab, *P < 0.05 versus −hRAGEab.

Article Snippet: Afterwards, human podocytes were lysed, protein concentration was assessed by Pierce BCA Protein Assay Kit (Thermo Scientific, Langenselbold, Germany) and after being boiled in sodium dodecyl sulphate (SDS), sample buffer 5–20 μg of lysate was separated by 8–12% SDS– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane.

Techniques: Staining, Incubation, Control, Quantitation Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Human RAGE antibody protects against AGE-mediated podocyte dysfunction.

doi: 10.1093/ndt/gfs005

Figure Lengend Snippet: Figure 4 Migration. Migration assay to investigate the effect of ±hRAGEab after 48 h of incubation on podocyte motility in the control, PDF and GDP group, phase contrast microscopy, scale bar represents 200 μm. *P < 0.05 versus −hRAGEab, #P < 0.05 versus control −hRAGEab.

Article Snippet: Afterwards, human podocytes were lysed, protein concentration was assessed by Pierce BCA Protein Assay Kit (Thermo Scientific, Langenselbold, Germany) and after being boiled in sodium dodecyl sulphate (SDS), sample buffer 5–20 μg of lysate was separated by 8–12% SDS– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane.

Techniques: Migration, Incubation, Control, Microscopy

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Human RAGE antibody protects against AGE-mediated podocyte dysfunction.

doi: 10.1093/ndt/gfs005

Figure Lengend Snippet: Figure 5 Apoptosis. Analysis of apoptosis in podocytes to investigate the effect of ±hRAGEab after 48 h of incubation in the control, PDF and GDP group. Apoptotic cells were detected by counting the number of fragmented nuclei (A) and by immunofluorescence staining of caspase 3 (B). ***P < 0.001 versus −hRAGEab, **P < 0.01 versus −hRAGEab, *P < 0.05 versus −hRAGEab.

Article Snippet: Afterwards, human podocytes were lysed, protein concentration was assessed by Pierce BCA Protein Assay Kit (Thermo Scientific, Langenselbold, Germany) and after being boiled in sodium dodecyl sulphate (SDS), sample buffer 5–20 μg of lysate was separated by 8–12% SDS– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane.

Techniques: Incubation, Control, Staining

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Human RAGE antibody protects against AGE-mediated podocyte dysfunction.

doi: 10.1093/ndt/gfs005

Figure Lengend Snippet: Figure 6 Working model. Proposed mechanism of PDF- and GDP- induced podocyte damage.

Article Snippet: Afterwards, human podocytes were lysed, protein concentration was assessed by Pierce BCA Protein Assay Kit (Thermo Scientific, Langenselbold, Germany) and after being boiled in sodium dodecyl sulphate (SDS), sample buffer 5–20 μg of lysate was separated by 8–12% SDS– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane.

Techniques: