Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 3. Validation of primary podocytes isolated from fawn-hooded hypertensive (FHH) and FHH.Add3 transgenic rats. A: representative images of freshly isolated glomeruli on day 0 (D0), cobblestone-like undifferentiated podocytes outgrowth from the decapsulated glomerulus on D5, and fully dif- ferentiated arborized podocytes on D10. Images were captured using a Lionheart FX Automated Microscope with 10 objective (total magnification of 350). B: representative images of nephrin-, synaptopodin (SYNPO)-, and podocin-positive primary podocytes by immunocytochemistry. C, top: repre- sentative images of c-adducin (ADD3) expression and distribution in primary podocytes. Podocytes were isolated from 6–9 male rats in each strain. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 60 oil immersion objective and a 3 digi- tal zoom (total magnification of 3,960). C, bottom: representative images and quantitation of ADD3 expression in podocytes by Western blot. BP, block- ing peptide; M, molecular mass. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Biomarker Discovery, Isolation, Transgenic Assay, Microscopy, Immunocytochemistry, Expressing, Inverted Microscopy, Quantitation Assay, Western Blot, Blocking Assay

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 4. Validation of conditionally immortalized mouse podocytes and the efficiency of knockdown of the expression of c-adducin (ADD3) with Add3 Dicer-substrate short interfering RNA (DsiRNA). A: representative images of nephrin-, synaptopodin (SYNPO)-, and podocin-positive conditionally immor- talized mouse podocytes by immunocytochemistry. B and C: representative images and quantitation of ADD3 immunostaining (B) and Western blots (C) of the efficiency of knockdown of the expression of ADD3 in conditionally immortalized mouse podocytes 36 h posttransfection with Add3 DsiRNA. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective (total magnification of 440). Experiments were repeated 4–5 times in triplicate in each experiment. Mean values ± SE are presented. P < 0.05 from the corresponding val- ues in NC1 negative controls (Ctrl).

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Biomarker Discovery, Knockdown, Expressing, Small Interfering RNA, Immunocytochemistry, Quantitation Assay, Immunostaining, Western Blot, Inverted Microscopy

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 5. Downregulation of c-adducin (ADD3) enhances F-actin net formation in podocytes. A: representative images (left) and quantitation (right) of im- munostaining for comparison of the expression and distribution of F/G-actin and the F/G-actin ratio in Add3 Dicer-substrate short interfering RNA (DsiRNA) and NC1 scrambled siRNA transfected immortalized mouse podocytes B: representative images (left) and quantitation (right) of immunostaining for comparison of the expression and distribution of F/G-actin and F/G-actin ratio in primary podocytes isolated from FHH and FHH.Add3 transgenic rats. C: representative images of immunostaining for comparison of the expression and distribution of ARPC1B (left) and quantitation with Western blots (right) in primary podocytes isolated from male FHH and FHH.Add3 transgenic rats. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 60 oil immersion objective and a 3 digital zoom (total magnification of 3,960). Experiments using condition- ally immortalized mouse podocytes were repeated 3 times in triplicate in each experiment. Podocytes were isolated from 6–9 male rats in each strain. ARPC1B, actin-related protein 2/3 complex subunit 1B. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats or NC1 negative controls (Ctrl).

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Quantitation Assay, Comparison, Expressing, Small Interfering RNA, Transfection, Immunostaining, Isolation, Transgenic Assay, Western Blot, Inverted Microscopy

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 6. Reduced c-adducin (ADD3) expression alters the Rho family of small GTPases levels in podocytes in fawn-hooded hypertensive (FHH) rats. A: representative images (left) and quantitation (right) of synaptopodin (SYNPO) immunostaining in the glo- meruli of male FHH and FHH.Add3 rats. Six sections per rat and 30 glomeruli per section were examined; n = 6 rats per group. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective and 1 digi- tal zoom (left, total magnification of 440) and 4.5 digital zoom (right, total magnification of 1,980). B: representative images (left) and quantitation (right) of SYNPO and RhoA expression in the renal cortex of male FHH and FHH.Add3 rats. C: representative images (left) and quantitation (right) of SYNPO and RhoA expression in the primary podocytes isolated from male FHH and FHH.Add3 rats. D: representa- tive images (left) and quantitation (right) of Rac1 and CDC42 expression in the renal cortex of male FHH and FHH.Add3 rats. E: representative images (left) and quantitation (right) of Rac1 and CDC42 expres- sion in primary podocytes isolated from male FHH and FHH.Add3 rats. Mean values ± SE are presented; n = 6–9 rats in each strain. P < 0.05 from the corre- sponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Expressing, Quantitation Assay, Immunostaining, Inverted Microscopy, Isolation

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 7. Reduced c-adducin (ADD3) expression attenu- ates slit diaphragm protein expression in fawn-hooded hypertensive (FHH) rats. Validation of the nephrin anti- body by Western blot in the renal cortex of male FHH and FHH.Add3 rats is shown. A and B: immunostaining using conditionally immortalized mouse podocytes (A) with and without the blocking peptide (BP; B). M, molec- ular mass. C: representative images (left) and quantita- tion (right) of nephrin immunostaining in the glomeruli of male FHH compared with FHH.Add3 rats. Six sections per rat and 30 glomeruli per section were examined; n = 6 rats per group. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective and 1 digital zoom (left, total magnification of 440) and 4.5 digital zoom (right, total magnification of 1,980). D: represen- tative images (left) and quantitation (right) of nephrin im- munostaining in primary podocytes of FHH compared with FHH.Add3 rats. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 60 oil immersion objective and a 3 digital zoom (total magnification of 3,960). E: representative images (left) and quantitation (right) of nephrin and podocin expression by Western blot in the renal cortex of male FHH and FHH.Add3 rats. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Expressing, Biomarker Discovery, Western Blot, Immunostaining, Blocking Assay, Inverted Microscopy, Quantitation Assay

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 8. Reduced c-adducin (ADD3) expres- sion affects focal adhesion integrins in fawn- hooded hypertensive (FHH) rats. A: representa- tive images (left) and quantitation (right) of integrin-a3 (ITGA30) immunostaining in the glo- meruli obtained from male FHH compared with FHH.Add3 rats. Six sections per rat and 30 glo- meruli per section were examined; n = 6 rats per group. B: representative images (left) and quantitation (right) of integrin-a3 (ITGB1) expres- sion in the renal cortex of male FHH and FHH. Add3 rats. Images in A and B were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective and 1 digital zoom (left, total magni- fication of 440) and 4.5 digital zoom (right, total magnification of 1,980). C: representative images (left) and quantitation (right) of ITGA3 and ITGB1 expression in the glomeruli of male FHH and FHH.Add3 rats. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Quantitation Assay, Immunostaining, Inverted Microscopy, Expressing