Structured Review

Santa Cruz Biotechnology synaptopodin
Effects of conditioned MSCs on expression of <t>synaptopodin</t> and fibronectin in podocytes. Podocytes were incubated without or with 15 ng/ml TGF-β1, conditioned MSCs and/or ascorbic acid for 72 hrs. Cell lysates were analysed with Western blotting. ( A ) Representative Western blot and bar graph analysis of relative protein level of synaptopodin, which were normalized to control. ( B ) Bar graph analysis of quantitative PCR analysis of relative fibronectin expression to GAPDH normalized to control. * P
Synaptopodin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/synaptopodin/product/Santa Cruz Biotechnology
Average 85 stars, based on 1 article reviews
Price from $9.99 to $1999.99
synaptopodin - by Bioz Stars, 2022-05
85/100 stars

Images

1) Product Images from "Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation"

Article Title: Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2012.01610.x

Effects of conditioned MSCs on expression of synaptopodin and fibronectin in podocytes. Podocytes were incubated without or with 15 ng/ml TGF-β1, conditioned MSCs and/or ascorbic acid for 72 hrs. Cell lysates were analysed with Western blotting. ( A ) Representative Western blot and bar graph analysis of relative protein level of synaptopodin, which were normalized to control. ( B ) Bar graph analysis of quantitative PCR analysis of relative fibronectin expression to GAPDH normalized to control. * P
Figure Legend Snippet: Effects of conditioned MSCs on expression of synaptopodin and fibronectin in podocytes. Podocytes were incubated without or with 15 ng/ml TGF-β1, conditioned MSCs and/or ascorbic acid for 72 hrs. Cell lysates were analysed with Western blotting. ( A ) Representative Western blot and bar graph analysis of relative protein level of synaptopodin, which were normalized to control. ( B ) Bar graph analysis of quantitative PCR analysis of relative fibronectin expression to GAPDH normalized to control. * P

Techniques Used: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction

2) Product Images from "Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)"

Article Title: Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Journal: Genes & Diseases

doi: 10.1016/j.gendis.2018.04.003

The imPOD cells express podocyte markers . The imPOD and parental tsPC (at 33 °C + 100U/ml γ-interferon) cells were seeded at subconfluence and stained with antibodies against the podocyte specific marker (WT-1) and Synaptopodin ( A ), as well as the slit diaphragm complex related markers (Nephrin, Tubulin and Vinculin) ( B ). Stains without primary antibodies were used as negative controls. Cell nuclei were counter-stained with DAPI. Representative images are shown.
Figure Legend Snippet: The imPOD cells express podocyte markers . The imPOD and parental tsPC (at 33 °C + 100U/ml γ-interferon) cells were seeded at subconfluence and stained with antibodies against the podocyte specific marker (WT-1) and Synaptopodin ( A ), as well as the slit diaphragm complex related markers (Nephrin, Tubulin and Vinculin) ( B ). Stains without primary antibodies were used as negative controls. Cell nuclei were counter-stained with DAPI. Representative images are shown.

Techniques Used: Staining, Marker

3) Product Images from "Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria"

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

Journal: Scientific Reports

doi: 10.1038/s41598-017-00564-3

Efficient and specific deletion of Shp2 in podocytes. ( a ) Genomic DNA was extracted from tissues (as indicated) of control (Ctrl) and pod-Shp2 knockout (KO) mice. Deletion of the floxed allele was detected by PCR, and GAPDH served as a loading control. ( b ) Immunoblots of Shp2 protein expression in isolated primary podocytes, adipose (epididymal fat), liver and muscle from Ctrl and KO mice. Tubulin and synaptopodin (Synpo, for podocytes) presented as loading controls. ( c ) Immunostaining of Shp2 (green) and synaptopodin (red) in kidney sections from Ctrl and KO mice. Scale bar: 50 µm.
Figure Legend Snippet: Efficient and specific deletion of Shp2 in podocytes. ( a ) Genomic DNA was extracted from tissues (as indicated) of control (Ctrl) and pod-Shp2 knockout (KO) mice. Deletion of the floxed allele was detected by PCR, and GAPDH served as a loading control. ( b ) Immunoblots of Shp2 protein expression in isolated primary podocytes, adipose (epididymal fat), liver and muscle from Ctrl and KO mice. Tubulin and synaptopodin (Synpo, for podocytes) presented as loading controls. ( c ) Immunostaining of Shp2 (green) and synaptopodin (red) in kidney sections from Ctrl and KO mice. Scale bar: 50 µm.

Techniques Used: Knock-Out, Mouse Assay, Polymerase Chain Reaction, Western Blot, Expressing, Isolation, Immunostaining

4) Product Images from "Myosin-1c promotes E-cadherin tension and force-dependent recruitment of α-actinin to the epithelial cell junction"

Article Title: Myosin-1c promotes E-cadherin tension and force-dependent recruitment of α-actinin to the epithelial cell junction

Journal: Journal of Cell Science

doi: 10.1242/jcs.211334

Myosin-1c colocalizes with F-actin and a tension-sensitive α-actinin-4 protein on the lateral junction in polarized epithelial monolayer. (A) OS-SIM showing immunofluorescence of myosin-1c (M1c), synaptopodin (Syp, synpo), and α-actinin-4 (A4) at the apical ( z =0 µm), the sub-apical ( z =0.4 µm) and the lateral ( z =2.2 µm) junctions. For each z -image, the top panel shows the x-z , y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of six independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (B) Merge images of the representative linear junction shown in A. The Pearson's correlation coefficients between myoin-1c, synaptopodin or α-actinin-4 at the junctional vertices (white circle) and linear junctions (white lozenge) are represented by Rv and Rj, respectively. Correlations were calculated from 500–1500 pixels per protein per z -image in one set of data. Six sets of data were analyzed and a representative set of data is shown. Scale bar: 2 µm. (C) OS-SIM showing immunofluorescence of myosin-1c, actin (phalloidin, Phal), and myosin IIB (MIIB) at the sub-apical ( z =1 µm) and the lateral ( z =2.6 and 4.2 µm) junctions. For each z -image, the top panel shows the x-z, y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of three independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (D) Merge images of the representative linear junction shown in C. The Pearson's correlation coefficient ( R ) between two proteins at the linear junction was calculated from 1000–2000 pixels per protein per z -image for one set of data. Data set is representative of six sets of data from one experiment out of three independent experiments. Scale bar: 2 µm.
Figure Legend Snippet: Myosin-1c colocalizes with F-actin and a tension-sensitive α-actinin-4 protein on the lateral junction in polarized epithelial monolayer. (A) OS-SIM showing immunofluorescence of myosin-1c (M1c), synaptopodin (Syp, synpo), and α-actinin-4 (A4) at the apical ( z =0 µm), the sub-apical ( z =0.4 µm) and the lateral ( z =2.2 µm) junctions. For each z -image, the top panel shows the x-z , y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of six independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (B) Merge images of the representative linear junction shown in A. The Pearson's correlation coefficients between myoin-1c, synaptopodin or α-actinin-4 at the junctional vertices (white circle) and linear junctions (white lozenge) are represented by Rv and Rj, respectively. Correlations were calculated from 500–1500 pixels per protein per z -image in one set of data. Six sets of data were analyzed and a representative set of data is shown. Scale bar: 2 µm. (C) OS-SIM showing immunofluorescence of myosin-1c, actin (phalloidin, Phal), and myosin IIB (MIIB) at the sub-apical ( z =1 µm) and the lateral ( z =2.6 and 4.2 µm) junctions. For each z -image, the top panel shows the x-z, y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of three independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (D) Merge images of the representative linear junction shown in C. The Pearson's correlation coefficient ( R ) between two proteins at the linear junction was calculated from 1000–2000 pixels per protein per z -image for one set of data. Data set is representative of six sets of data from one experiment out of three independent experiments. Scale bar: 2 µm.

Techniques Used: Immunofluorescence

Myosin-1c knockdown abolishes junction recruitment of α-actinin-4 and weakens cell-cell cohesion. (A) OS-SIM images of myosin-1c (Myo1c), α-catenin, p120-catenin and E-cadherin at the apical plane of heterogeneous myosin-1c knockdown (KD) cell monolayers. Data are representative of 12 sets of images from one experiment out of three independent experiments. (B) OS-SIM images of myosin-1c, β-catenin, synaptopodin (synpo) and α-actinin-4 at the apical plane of heterogeneous myosin-1c knockdown cell monolayers. Data are representative of 12 sets of images from one experiment out of three different independent experiments. (C) Quantification of junctional myosin-1c, α-catenin, p120-catenin, E-cadherin, β-catenin, synaptopodin and α-actinin-4 immunofluorescence in wild-type (WT) and myosin-1c knockdown (Myo1cKD) cells. Bar graphs show the normalized mean±s.e.m. intensity of 24 junctions. P
Figure Legend Snippet: Myosin-1c knockdown abolishes junction recruitment of α-actinin-4 and weakens cell-cell cohesion. (A) OS-SIM images of myosin-1c (Myo1c), α-catenin, p120-catenin and E-cadherin at the apical plane of heterogeneous myosin-1c knockdown (KD) cell monolayers. Data are representative of 12 sets of images from one experiment out of three independent experiments. (B) OS-SIM images of myosin-1c, β-catenin, synaptopodin (synpo) and α-actinin-4 at the apical plane of heterogeneous myosin-1c knockdown cell monolayers. Data are representative of 12 sets of images from one experiment out of three different independent experiments. (C) Quantification of junctional myosin-1c, α-catenin, p120-catenin, E-cadherin, β-catenin, synaptopodin and α-actinin-4 immunofluorescence in wild-type (WT) and myosin-1c knockdown (Myo1cKD) cells. Bar graphs show the normalized mean±s.e.m. intensity of 24 junctions. P

Techniques Used: Immunofluorescence

5) Product Images from "Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation"

Article Title: Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn955

Differentiation of MPC5 cells. (A) Mouse MPC5 cells were differentiated for the times indicated at top. The cells were then fixed and subject to immunofluorescence with either anti-tubulin or anti-synaptopodin antibodies (both in green), counterstaining with Hoechst. (B) MPC5 cells were differentiated for the number of days indicated and total RNA prepared. Semi-quantitative RT-PCR was performed to detect WT1, CSF1, PDGFA, Bak, c-myc, Podocalyxin and GAPDH.
Figure Legend Snippet: Differentiation of MPC5 cells. (A) Mouse MPC5 cells were differentiated for the times indicated at top. The cells were then fixed and subject to immunofluorescence with either anti-tubulin or anti-synaptopodin antibodies (both in green), counterstaining with Hoechst. (B) MPC5 cells were differentiated for the number of days indicated and total RNA prepared. Semi-quantitative RT-PCR was performed to detect WT1, CSF1, PDGFA, Bak, c-myc, Podocalyxin and GAPDH.

Techniques Used: Immunofluorescence, Quantitative RT-PCR

6) Product Images from "RNA-binding proteins tristetraprolin and human antigen R are novel modulators of podocyte injury in diabetic kidney disease"

Article Title: RNA-binding proteins tristetraprolin and human antigen R are novel modulators of podocyte injury in diabetic kidney disease

Journal: Cell Death & Disease

doi: 10.1038/s41419-020-2630-x

GSK-3β inhibition alleviated podocyte injury and inflammation in STZ-induced diabetic mice (C57BL/6J mice). a Effect of STZ and TDZD-8 treatment on blood glucose (left) and urine albumin adjusted by creatinine (uAcr) concentrations. b PAS staining and electron microscopy showed glomerular injury caused by diabetes. Marked foot process effacement and basement membrane thickening are shown (black arrows). Morphometric analyses of foot processes per 3 µm glomerular basement membrane and the thickness of the glomerular basement membrane was observed by electron microscopy observation (right). Scale bar: 20 μm for PAS staining; 1 μm for electron microscopy. c - e Dual-color fluorescence staining of kidney sections for podocin and claudin-1 ( c ), synaptopodin and IL-17 ( d ), and synaptopodin and CD80 ( e ). f , g Lysates of isolated glomeruli were analyzed by immunoblotting for expression of podocytes marker proteins and podocytes injury proteins. GAPDH served as a loading control. h. Lysates of glomeruli were analyzed by immunoblotting for expression of S9-phosphorylated GSK-3β, total GSK-3β, and GAPDH. Data are expressed as the mean ± SD. * p
Figure Legend Snippet: GSK-3β inhibition alleviated podocyte injury and inflammation in STZ-induced diabetic mice (C57BL/6J mice). a Effect of STZ and TDZD-8 treatment on blood glucose (left) and urine albumin adjusted by creatinine (uAcr) concentrations. b PAS staining and electron microscopy showed glomerular injury caused by diabetes. Marked foot process effacement and basement membrane thickening are shown (black arrows). Morphometric analyses of foot processes per 3 µm glomerular basement membrane and the thickness of the glomerular basement membrane was observed by electron microscopy observation (right). Scale bar: 20 μm for PAS staining; 1 μm for electron microscopy. c - e Dual-color fluorescence staining of kidney sections for podocin and claudin-1 ( c ), synaptopodin and IL-17 ( d ), and synaptopodin and CD80 ( e ). f , g Lysates of isolated glomeruli were analyzed by immunoblotting for expression of podocytes marker proteins and podocytes injury proteins. GAPDH served as a loading control. h. Lysates of glomeruli were analyzed by immunoblotting for expression of S9-phosphorylated GSK-3β, total GSK-3β, and GAPDH. Data are expressed as the mean ± SD. * p

Techniques Used: Inhibition, Mouse Assay, Staining, Electron Microscopy, Fluorescence, Isolation, Expressing, Marker

Modulation of TTP and HuR expressions by TDZD-8 treatment in STZ-treated mice. a Immunoblotting of glomerular lysates to detect TTP and HuR expressions. b Dual-color fluorescence staining for synaptopodin (red) and TTP (green). c Dual-color fluorescence staining for WT-1 (green) and HuR (red) showed nuclear accumulation of HuR in glomerular podocytes in STZ-treated (C57BL/6J wildtype) mice. TDZD-8 injection reduced the nuclear accumulation of HuR. d Numbers of WT-1-positive (top) and HuR/WT-1-positive (bottom) podocytes per glomerulus in kidney specimens. Data are expressed as the mean ± SD; six mice in each group; n = 6 for each experiment. * p
Figure Legend Snippet: Modulation of TTP and HuR expressions by TDZD-8 treatment in STZ-treated mice. a Immunoblotting of glomerular lysates to detect TTP and HuR expressions. b Dual-color fluorescence staining for synaptopodin (red) and TTP (green). c Dual-color fluorescence staining for WT-1 (green) and HuR (red) showed nuclear accumulation of HuR in glomerular podocytes in STZ-treated (C57BL/6J wildtype) mice. TDZD-8 injection reduced the nuclear accumulation of HuR. d Numbers of WT-1-positive (top) and HuR/WT-1-positive (bottom) podocytes per glomerulus in kidney specimens. Data are expressed as the mean ± SD; six mice in each group; n = 6 for each experiment. * p

Techniques Used: Mouse Assay, Fluorescence, Staining, Injection

Imbalanced expression of TTP and HuR in kidney tissues of DKD patients. a Urine albumin excretion adjusted by urine creatinine (uACR) levels (top) and blood glucose levels (bottom) of DM and DKD patients. b PAS staining and electron microscopy of kidney tissues. Left, foot process effacement and basement membrane thickening are denoted by black arrows. Right, morphometric observation of numbers of foot processes per 3 µm glomerular basement membrane (GBM) and the thickness of GBM. Scale bars: 50 μm for PAS staining; 1 μm for electron microscopy. c Dual-color fluorescence staining for podocin and claudin-1 in kidney sections from DKD patients. d Dual-color immunofluorescence for synaptopodin and IL-17, white arrows indicate the colocalization of synaptopodin and IL-17. e Immunofluorescence staining for WT-1 and HuR demonstrating the nuclear accumulation of HuR in glomerular podocytes. f Numbers of WT-1-positive (top) and percentage of HuR/WT-1-positive (bottom) podocytes per glomerulus. g Immunohistochemical staining in kidney tissues for TTP. h Relative intensity of TTP positive staining per glomerulus. i Linear regression analysis showed a negative correlation between relative TTP expression (left) and uACR, and a positive correlation between both HuR and WT-1 positive podocytes (right) and uACR ( n = 15). Data are expressed as mean ± SD. n = 6 for each separate experiment. * p
Figure Legend Snippet: Imbalanced expression of TTP and HuR in kidney tissues of DKD patients. a Urine albumin excretion adjusted by urine creatinine (uACR) levels (top) and blood glucose levels (bottom) of DM and DKD patients. b PAS staining and electron microscopy of kidney tissues. Left, foot process effacement and basement membrane thickening are denoted by black arrows. Right, morphometric observation of numbers of foot processes per 3 µm glomerular basement membrane (GBM) and the thickness of GBM. Scale bars: 50 μm for PAS staining; 1 μm for electron microscopy. c Dual-color fluorescence staining for podocin and claudin-1 in kidney sections from DKD patients. d Dual-color immunofluorescence for synaptopodin and IL-17, white arrows indicate the colocalization of synaptopodin and IL-17. e Immunofluorescence staining for WT-1 and HuR demonstrating the nuclear accumulation of HuR in glomerular podocytes. f Numbers of WT-1-positive (top) and percentage of HuR/WT-1-positive (bottom) podocytes per glomerulus. g Immunohistochemical staining in kidney tissues for TTP. h Relative intensity of TTP positive staining per glomerulus. i Linear regression analysis showed a negative correlation between relative TTP expression (left) and uACR, and a positive correlation between both HuR and WT-1 positive podocytes (right) and uACR ( n = 15). Data are expressed as mean ± SD. n = 6 for each separate experiment. * p

Techniques Used: Expressing, Staining, Electron Microscopy, Fluorescence, Immunofluorescence, Immunohistochemistry

Altered expression of TTP and HuR and podocyte injury in db / db mice. a Blood glucose and urine albumin adjusted by creatinine (uAcr) concentrations. b PAS staining and electron microscopy revealed the glomerular injury in db / db mice. Left, PAS staining showed matrix accumulation in db / db mice and electron microscopy indicated marked foot process effacement (black arrows). Right, morphometric analysis of numbers of foot processes per 3 µm glomerular basement membrane (GBM) and the thickness of GBM by electron microscopy. Scale bar: 20μm for PAS staining; 1 μm for electron microscopy. c , d Dual-color fluorescence staining of kidney sections to detect podocin, claudin-1, synaptopodin, and IL-17. e Dual-color immunofluorescence for WT-1 (red) and HuR (green). f Numbers of podocytes positive for WT-1 per glomerulus as the means of six glomeruli (top) and podocytes positive for both HuR and WT-1 positive staining in each glomerulus as the means of six glomeruli (bottom). g Dual-color immunofluorescence staining for synaptopodin (green) and TTP (red). h Immunoblotting of glomerular extracts to detect podocyte marker proteins, claudin-1 and IL-17 expression. i Immunoblotting of glomerular extracts to detect TTP and HuR expressions (top). The ratio of TTP/HuR expression in different groups was analyzed (bottom). Quantified immunoblot data are expressed as the mean ± SD; six mice in each group; n = 6 for each separate experiment. * p
Figure Legend Snippet: Altered expression of TTP and HuR and podocyte injury in db / db mice. a Blood glucose and urine albumin adjusted by creatinine (uAcr) concentrations. b PAS staining and electron microscopy revealed the glomerular injury in db / db mice. Left, PAS staining showed matrix accumulation in db / db mice and electron microscopy indicated marked foot process effacement (black arrows). Right, morphometric analysis of numbers of foot processes per 3 µm glomerular basement membrane (GBM) and the thickness of GBM by electron microscopy. Scale bar: 20μm for PAS staining; 1 μm for electron microscopy. c , d Dual-color fluorescence staining of kidney sections to detect podocin, claudin-1, synaptopodin, and IL-17. e Dual-color immunofluorescence for WT-1 (red) and HuR (green). f Numbers of podocytes positive for WT-1 per glomerulus as the means of six glomeruli (top) and podocytes positive for both HuR and WT-1 positive staining in each glomerulus as the means of six glomeruli (bottom). g Dual-color immunofluorescence staining for synaptopodin (green) and TTP (red). h Immunoblotting of glomerular extracts to detect podocyte marker proteins, claudin-1 and IL-17 expression. i Immunoblotting of glomerular extracts to detect TTP and HuR expressions (top). The ratio of TTP/HuR expression in different groups was analyzed (bottom). Quantified immunoblot data are expressed as the mean ± SD; six mice in each group; n = 6 for each separate experiment. * p

Techniques Used: Expressing, Mouse Assay, Staining, Electron Microscopy, Fluorescence, Immunofluorescence, Marker

7) Product Images from "CASK, the Soluble Glomerular Permeability Factor, Is Secreted by Macrophages in Patients With Recurrent Focal and Segmental Glomerulo—Sclerosis"

Article Title: CASK, the Soluble Glomerular Permeability Factor, Is Secreted by Macrophages in Patients With Recurrent Focal and Segmental Glomerulo—Sclerosis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00875

Cytoskeleton alterations induced by exosomes from M1 or M2 macrophages. Exosomes from M1 and M2 macrophages were incubated with human podocytes at concentrations of 10 and 40 μg/ml. Immunostaining was performed with phalloidin (green) and anti-synaptopodin antibody (red). Analysis was performed by fluorescence microscopy with a ×63 objective.
Figure Legend Snippet: Cytoskeleton alterations induced by exosomes from M1 or M2 macrophages. Exosomes from M1 and M2 macrophages were incubated with human podocytes at concentrations of 10 and 40 μg/ml. Immunostaining was performed with phalloidin (green) and anti-synaptopodin antibody (red). Analysis was performed by fluorescence microscopy with a ×63 objective.

Techniques Used: Incubation, Immunostaining, Fluorescence, Microscopy

8) Product Images from "Fine-tuning of NFκB by Glycogen Synthase Kinase 3β directs the fate of glomerular podocytes upon injury"

Article Title: Fine-tuning of NFκB by Glycogen Synthase Kinase 3β directs the fate of glomerular podocytes upon injury

Journal: Kidney international

doi: 10.1038/ki.2014.428

Fine tuning of NFκB activity by GSK3β in LPS induced experimental podocytopathy Mice were treated as described in Figure 6 . (A and B) Homogenates of glomeruli isolated from mouse kidneys were prepared and subjected to immunoblot analysis for indicated molecules. Nuclear protein fractions were prepared from isolated glomeruli and subjected to immunoblot analysis for nuclear β-catenin (n-β-catenin) or for nuclear protein histone (n-Histone H3), which served as loading controls. (C) Representative micrographs of laser scanning confocal microscopy of fluorescent immunohistochemistry staining demonstrate an enhanced glomerular expression of B7-1, MCP-1, and Bcl-xL in kidney specimens from LPS injured mice. Note that the staining of B7-1, MCP-1 and Bcl-xL were largely detected in glomerular cells positive for podocyte specific markers, like synaptopodin and WT-1, suggesting a pattern of podocyte distribution. In the enlarged view of the boxed area, B7-1 was mainly found to colocalize with synaptopodin, MCP-1 mostly detected in cytoplasm of WT-1 positive glomerular cells, and Bcl-xL largely found to be external to synaptopodin, all indicative of a pattern of podocyte distribution. Lithium or TDZD-8 therapy attenuated the LPS induced glomerular expression of B7-1 and MCP-1, but barely affected the upregulated expression of Bcl-xL. TPCK treatment abolished the LPS elicited glomerular expression of all NFκB target molecules, including both podocytopathic ones, like MCP-1 and B7-1, and the prosurvival Bcl-xL. Bar=50µM.
Figure Legend Snippet: Fine tuning of NFκB activity by GSK3β in LPS induced experimental podocytopathy Mice were treated as described in Figure 6 . (A and B) Homogenates of glomeruli isolated from mouse kidneys were prepared and subjected to immunoblot analysis for indicated molecules. Nuclear protein fractions were prepared from isolated glomeruli and subjected to immunoblot analysis for nuclear β-catenin (n-β-catenin) or for nuclear protein histone (n-Histone H3), which served as loading controls. (C) Representative micrographs of laser scanning confocal microscopy of fluorescent immunohistochemistry staining demonstrate an enhanced glomerular expression of B7-1, MCP-1, and Bcl-xL in kidney specimens from LPS injured mice. Note that the staining of B7-1, MCP-1 and Bcl-xL were largely detected in glomerular cells positive for podocyte specific markers, like synaptopodin and WT-1, suggesting a pattern of podocyte distribution. In the enlarged view of the boxed area, B7-1 was mainly found to colocalize with synaptopodin, MCP-1 mostly detected in cytoplasm of WT-1 positive glomerular cells, and Bcl-xL largely found to be external to synaptopodin, all indicative of a pattern of podocyte distribution. Lithium or TDZD-8 therapy attenuated the LPS induced glomerular expression of B7-1 and MCP-1, but barely affected the upregulated expression of Bcl-xL. TPCK treatment abolished the LPS elicited glomerular expression of all NFκB target molecules, including both podocytopathic ones, like MCP-1 and B7-1, and the prosurvival Bcl-xL. Bar=50µM.

Techniques Used: Activity Assay, Mouse Assay, Isolation, Confocal Microscopy, Immunohistochemistry, Staining, Expressing

LPS injury triggers NFκB activation, elicits GSK3β overactivity and induces injury in cultured murine podocytes (A) Differentiated immortalized murine podocytes in culture were injured with varying doses of LPS (0, 1, 10, 20, 50 µg/ml) for 24 hours. Cell lysates were prepared for immunoblot analysis for indicated molecules, including TLR-4, phosphorylated GSK3β, phosphorylated RelA/p65 at S467, S536 and S276 residues, GAPDH and NFκB target molecules, such as B7-1, cathepsin L, and Bcl-xL; (B) Podocytes were treated with LPS (20 µg/ml) and harvested at indicated time. Cell lysates were processed for immunoblot analysis for indicated molecules; (C) Podocytes were treated with LPS (20 µg/ml) or equal volume of phosphate-buffered saline (PBS) for 24 h and fixed for fluorescent staining. Representative micrographs of laser scanning confocal fluorescence microscopy show nuclear translocation of NFκB RelA/p65, expression of B7-1 and synaptopodin (synpo), and phalloidin staining for F-actin in PBS or LPS treated podocytes. Bar=10µM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3β, Glycogen synthase kinase 3β; LPS, lipopolysaccharides; PBS, phosphate-buffered saline; TLR-4, toll like receptor-4.
Figure Legend Snippet: LPS injury triggers NFκB activation, elicits GSK3β overactivity and induces injury in cultured murine podocytes (A) Differentiated immortalized murine podocytes in culture were injured with varying doses of LPS (0, 1, 10, 20, 50 µg/ml) for 24 hours. Cell lysates were prepared for immunoblot analysis for indicated molecules, including TLR-4, phosphorylated GSK3β, phosphorylated RelA/p65 at S467, S536 and S276 residues, GAPDH and NFκB target molecules, such as B7-1, cathepsin L, and Bcl-xL; (B) Podocytes were treated with LPS (20 µg/ml) and harvested at indicated time. Cell lysates were processed for immunoblot analysis for indicated molecules; (C) Podocytes were treated with LPS (20 µg/ml) or equal volume of phosphate-buffered saline (PBS) for 24 h and fixed for fluorescent staining. Representative micrographs of laser scanning confocal fluorescence microscopy show nuclear translocation of NFκB RelA/p65, expression of B7-1 and synaptopodin (synpo), and phalloidin staining for F-actin in PBS or LPS treated podocytes. Bar=10µM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3β, Glycogen synthase kinase 3β; LPS, lipopolysaccharides; PBS, phosphate-buffered saline; TLR-4, toll like receptor-4.

Techniques Used: Activation Assay, Cell Culture, Staining, Fluorescence, Microscopy, Translocation Assay, Expressing

9) Product Images from "Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury"

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2016091021

Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2 Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α -actinin4, and nephrin as well as mesenchymal proteins desmin and α -SMA. Representative micrographs are shown. Scale bar, 10 μ m. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α -actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2 Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μ m (C) or 10 μ m (D). Quantification data are shown in the right panel. *** P
Figure Legend Snippet: Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2 Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α -actinin4, and nephrin as well as mesenchymal proteins desmin and α -SMA. Representative micrographs are shown. Scale bar, 10 μ m. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α -actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2 Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μ m (C) or 10 μ m (D). Quantification data are shown in the right panel. *** P

Techniques Used: Mouse Assay, Staining, Knock-Out, Expressing, Immunofluorescence, Isolation

10) Product Images from "CLIC5A, a component of the ezrin-podocalyxin complex in glomeruli, is a determinant of podocyte integrity"

Article Title: CLIC5A, a component of the ezrin-podocalyxin complex in glomeruli, is a determinant of podocyte integrity

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00030.2010

CLIC5A protein expression. A : confocal microscopy with anti-CLIC5 antiserum (red) and anti-Tie-2 antibodies (green) in human glomeruli. B : confocal microscopy with anti-CLIC5 antiserum (red) and anti-synaptopodin antibodies (green) in human glomeruli.
Figure Legend Snippet: CLIC5A protein expression. A : confocal microscopy with anti-CLIC5 antiserum (red) and anti-Tie-2 antibodies (green) in human glomeruli. B : confocal microscopy with anti-CLIC5 antiserum (red) and anti-synaptopodin antibodies (green) in human glomeruli.

Techniques Used: Expressing, Confocal Microscopy

11) Product Images from "Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition"

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.07.025

Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n  = 6 controls and 8 patients with HIVAN. ∗∗∗ P ÂÂ
Figure Legend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n  = 6 controls and 8 patients with HIVAN. ∗∗∗ P ÂÂ

Techniques Used: Expressing, Labeling, Fluorescence, Software

The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n  = 4 ( A and C–F ). ∗ P ÂÂ
Figure Legend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n  = 4 ( A and C–F ). ∗ P ÂÂ

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n  = 4. ∗∗∗ P ÂÂ
Figure Legend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n  = 4. ∗∗∗ P ÂÂ

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n  = 4 ( A and B ); n  = 3 ( E and F ). ∗ P ÂÂ
Figure Legend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n  = 4 ( A and B ); n  = 3 ( E and F ). ∗ P ÂÂ

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence

12) Product Images from "Lineage Specification of Parietal Epithelial Cells Requires β-Catenin/Wnt Signaling"

Article Title: Lineage Specification of Parietal Epithelial Cells Requires β-Catenin/Wnt Signaling

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2010121257

β -Catenin–deficient mice show abnormal glomeruli featuring parietal podocytes, parietal capillaries, and underdeveloped capillary tufts. Semithin sections (A and B) and electron microscopy (C–H) of adult β -catenin–deficient (lox/lox Pax8Cre) kidneys. (A) The outermost cortex is missing. Glomeruli show underdeveloped capillary tufts and are cystically widened. Juxtamedullary glomeruli mostly appeared normal. (B) The glomeruli have a normal tubular pole (TP). (C) Electron microscopy reveals that the parietal epithelial cells are parietal podocytes (PP) with foot processes extending and covering Bowman’s capsule. Capillaries (asterisks) are arranged on the outer aspect of Bowman's capsule adjacent to parietal podocytes. VP, visceral podocytes. (D) Parietal podocytes (PP) and foot processes at higher magnification. Large parietal capillaries (PC) have formed adjacent to the parietal podocytes. (E) Parietal podocytes form a filtration-like barrier consisting of three layers: fenestrated endothelial cells (FE), a parietal basement membrane (BM), and foot processes (FP). The filtration slits between the foot processes are bridged by a slit membrane (arrows), and the endothelial fenestrae are bridged by diaphragms (arrowheads). (F) Parietal capillaries (asterisks) associated with parietal podocytes; one capillary bulges into Bowman's space (BS). (G) Bulging capillary with a well developed capillary neck (boxed area enlarged in I). (H) A schematic draft of the image of (G). Parietal podocytes (blue; PP) are covering the surface of a bulging parietal capillary (red; PC). Parietal basement membrane is dark blue. BS, Bowman’s space. (I) At the neck, the peripheral endothelial basement membrane reflects into the parietal basement membrane (white arrows); the bridging portion of the endothelium is underlain only by a thin and fragmented basement membrane (arrowheads). The cells within the neck region have many actin-filled processes (black arrows) that are connected to the basement membrane at the turning points. (J and K) Both visceral and parietal podocytes (arrows) stain positive for WT1. (L and M) Both visceral and parietal podocytes (arrows) stain positive for VEGF. (N and O) Double staining for synaptopodin (red) and WT1 (green). Synaptopodin in control kidneys is exclusively expressed in visceral podocytes. Parietal epithelial cells are negative for synaptopodin. In β -catenin–deficient mice, synaptopodin is expressed in both visceral and parietal podocytes (arrow). Original magnifications: ×125 in A; ×500 in B, J, K, N, and O; ×570 in C; ×3400 in D; ×40,000 in E; ×2000 in F; ×6300 in G; ×12,500 in I; ×750 in L and M.
Figure Legend Snippet: β -Catenin–deficient mice show abnormal glomeruli featuring parietal podocytes, parietal capillaries, and underdeveloped capillary tufts. Semithin sections (A and B) and electron microscopy (C–H) of adult β -catenin–deficient (lox/lox Pax8Cre) kidneys. (A) The outermost cortex is missing. Glomeruli show underdeveloped capillary tufts and are cystically widened. Juxtamedullary glomeruli mostly appeared normal. (B) The glomeruli have a normal tubular pole (TP). (C) Electron microscopy reveals that the parietal epithelial cells are parietal podocytes (PP) with foot processes extending and covering Bowman’s capsule. Capillaries (asterisks) are arranged on the outer aspect of Bowman's capsule adjacent to parietal podocytes. VP, visceral podocytes. (D) Parietal podocytes (PP) and foot processes at higher magnification. Large parietal capillaries (PC) have formed adjacent to the parietal podocytes. (E) Parietal podocytes form a filtration-like barrier consisting of three layers: fenestrated endothelial cells (FE), a parietal basement membrane (BM), and foot processes (FP). The filtration slits between the foot processes are bridged by a slit membrane (arrows), and the endothelial fenestrae are bridged by diaphragms (arrowheads). (F) Parietal capillaries (asterisks) associated with parietal podocytes; one capillary bulges into Bowman's space (BS). (G) Bulging capillary with a well developed capillary neck (boxed area enlarged in I). (H) A schematic draft of the image of (G). Parietal podocytes (blue; PP) are covering the surface of a bulging parietal capillary (red; PC). Parietal basement membrane is dark blue. BS, Bowman’s space. (I) At the neck, the peripheral endothelial basement membrane reflects into the parietal basement membrane (white arrows); the bridging portion of the endothelium is underlain only by a thin and fragmented basement membrane (arrowheads). The cells within the neck region have many actin-filled processes (black arrows) that are connected to the basement membrane at the turning points. (J and K) Both visceral and parietal podocytes (arrows) stain positive for WT1. (L and M) Both visceral and parietal podocytes (arrows) stain positive for VEGF. (N and O) Double staining for synaptopodin (red) and WT1 (green). Synaptopodin in control kidneys is exclusively expressed in visceral podocytes. Parietal epithelial cells are negative for synaptopodin. In β -catenin–deficient mice, synaptopodin is expressed in both visceral and parietal podocytes (arrow). Original magnifications: ×125 in A; ×500 in B, J, K, N, and O; ×570 in C; ×3400 in D; ×40,000 in E; ×2000 in F; ×6300 in G; ×12,500 in I; ×750 in L and M.

Techniques Used: Mouse Assay, Electron Microscopy, Filtration, Staining, Double Staining

13) Product Images from "The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury"

Article Title: The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2016111248

Calpain activation occurs in glomeruli of patients with FSGS. (A) Calpain activity was determined in the urine of patients with FSGS ( n =7) or healthy controls (control; n =5). (B) Protein was extracted from renal cortex samples, and a calpain activity assay was performed. Sections of the cortex were cut, and using an in situ zymography assay, (C) glomerular and (D) tubular calpain activity was determined. (E) Calpain activity in the section supernatant was measured and normalized to surface area of the section. (F) Western blot analysis was performed to determine the cortical expression of the calpain cleavage target Talin-1 (Talin-1–to– β -actin ratio: 1.22±0.17 in control versus 0.25±0.20 in FSGS samples). (G) Glomerular Talin-1 was costained with synaptopodin, and its expression was quantified. (H) Talin-1 was stained in combination with an in situ zymography for calpain activity in sections from patients with FSGS. (B–H) FSGS, n =3; control, n =5. * P
Figure Legend Snippet: Calpain activation occurs in glomeruli of patients with FSGS. (A) Calpain activity was determined in the urine of patients with FSGS ( n =7) or healthy controls (control; n =5). (B) Protein was extracted from renal cortex samples, and a calpain activity assay was performed. Sections of the cortex were cut, and using an in situ zymography assay, (C) glomerular and (D) tubular calpain activity was determined. (E) Calpain activity in the section supernatant was measured and normalized to surface area of the section. (F) Western blot analysis was performed to determine the cortical expression of the calpain cleavage target Talin-1 (Talin-1–to– β -actin ratio: 1.22±0.17 in control versus 0.25±0.20 in FSGS samples). (G) Glomerular Talin-1 was costained with synaptopodin, and its expression was quantified. (H) Talin-1 was stained in combination with an in situ zymography for calpain activity in sections from patients with FSGS. (B–H) FSGS, n =3; control, n =5. * P

Techniques Used: Activation Assay, Activity Assay, In Situ, Zymography, Western Blot, Expressing, Staining

Reduced calpastatin, increased Transient Receptor Potential channel C6 (TRPC6) expression, and enhanced calcineurin activation in patients with FSGS. (A) Representative pictures of costaining of the endogenous calpain inhibitor calpastatin and nephrin in healthy controls and patients with FSGS. (B) Calpastatin expression in podocytes was determined by quantifying the costaining of calpastatin and nephrin. (C) Cortical calcineurin activity, as a downstream calpain target, was determined as well as (D) cortical TRPC6 mRNA and (E) glomerular TRPC6 protein expression as quantified by immunofluorescence costained with synaptopodin. FSGS, n =3; control, n =5. * P
Figure Legend Snippet: Reduced calpastatin, increased Transient Receptor Potential channel C6 (TRPC6) expression, and enhanced calcineurin activation in patients with FSGS. (A) Representative pictures of costaining of the endogenous calpain inhibitor calpastatin and nephrin in healthy controls and patients with FSGS. (B) Calpastatin expression in podocytes was determined by quantifying the costaining of calpastatin and nephrin. (C) Cortical calcineurin activity, as a downstream calpain target, was determined as well as (D) cortical TRPC6 mRNA and (E) glomerular TRPC6 protein expression as quantified by immunofluorescence costained with synaptopodin. FSGS, n =3; control, n =5. * P

Techniques Used: Expressing, Activation Assay, Activity Assay, Immunofluorescence

Inhibition of the enhanced calpain activity in an animal model for human FSGS improves renal outcome. Wistar rats were injected with vehicle (control) or adriamycin (ADRIA) to induce ADRIA nephropathy, a model for human FSGS. Subsequently, animals were treated with vehicle or calpeptin for 6 weeks, after which they were euthanized. (A) Urinary calpain activity was determined. (B) Protein was isolated from renal cortex, and a calpain activity assay was performed. Sections of the cortex were cut, an in situ zymography assay was performed, and (C) glomerular and (D) tubular calpain activity was determined. (E) Western blot analysis was performed to determine the expression of the calpain cleavage target Talin-1 in cortex extracts of snap-frozen kidneys (Talin-1–to– β -actin ratios: control, 0.20; ADRIA, 0.01; ADRIA + calpeptin, 0.25; control + calpeptin, 0.35). (F) Glomerular Talin-1 was costained with synaptopodin, and its expression was quantified. (G) Talin-1 was stained in combination with an in situ zymography for calpain activity. (H) Cortical calcineurin activity, as a downstream calpain target, was determined. (I) Cortical expression of Transient Receptor Potential channel C6 (TRPC6) mRNA was determined as well as (J) glomerular TRPC6 protein expression costained with synaptopodin measured by immunofluorescence. * P
Figure Legend Snippet: Inhibition of the enhanced calpain activity in an animal model for human FSGS improves renal outcome. Wistar rats were injected with vehicle (control) or adriamycin (ADRIA) to induce ADRIA nephropathy, a model for human FSGS. Subsequently, animals were treated with vehicle or calpeptin for 6 weeks, after which they were euthanized. (A) Urinary calpain activity was determined. (B) Protein was isolated from renal cortex, and a calpain activity assay was performed. Sections of the cortex were cut, an in situ zymography assay was performed, and (C) glomerular and (D) tubular calpain activity was determined. (E) Western blot analysis was performed to determine the expression of the calpain cleavage target Talin-1 in cortex extracts of snap-frozen kidneys (Talin-1–to– β -actin ratios: control, 0.20; ADRIA, 0.01; ADRIA + calpeptin, 0.25; control + calpeptin, 0.35). (F) Glomerular Talin-1 was costained with synaptopodin, and its expression was quantified. (G) Talin-1 was stained in combination with an in situ zymography for calpain activity. (H) Cortical calcineurin activity, as a downstream calpain target, was determined. (I) Cortical expression of Transient Receptor Potential channel C6 (TRPC6) mRNA was determined as well as (J) glomerular TRPC6 protein expression costained with synaptopodin measured by immunofluorescence. * P

Techniques Used: Inhibition, Activity Assay, Animal Model, Injection, Isolation, In Situ, Zymography, Western Blot, Expressing, Staining, Immunofluorescence

14) Product Images from "Formation and optimization of three-dimensional organoids generated from urine-derived stem cells for renal function in vitro"

Article Title: Formation and optimization of three-dimensional organoids generated from urine-derived stem cells for renal function in vitro

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-020-01822-4

Expression of renal-specific markers in USCs and 3D organoids via RT-qPCR. a The expression level of specific proximal tubule AQP1. There was near none AQP1 expression in USCs, but three groups of organoids all had AQP1 expression. The AQP1 expression level of USC-organoids with kECM was between USC-organoids and renal organoids. b The expression level of kidney endocrine product EPO. Only USC-organoids with kECM and renal organoids had clear EPO expression. c The expression level of kidney glomerular marker Podocin. The Podocin expression level of USC-organoids with kECM was between USC-organoids and renal organoids. d The expression level of kidney glomerular marker Synaptopodin. The Synaptopodin expression level of USC-organoids with kECM was between USC-organoids and renal organoids. The AQP1, EPO, Podocin, and Synaptopodin expression level of USCs, USC-organoids, and USC-organoids with kECM were all significantly lower than renal organoids (three replicated samples for each group, data presented as mean ± SD)
Figure Legend Snippet: Expression of renal-specific markers in USCs and 3D organoids via RT-qPCR. a The expression level of specific proximal tubule AQP1. There was near none AQP1 expression in USCs, but three groups of organoids all had AQP1 expression. The AQP1 expression level of USC-organoids with kECM was between USC-organoids and renal organoids. b The expression level of kidney endocrine product EPO. Only USC-organoids with kECM and renal organoids had clear EPO expression. c The expression level of kidney glomerular marker Podocin. The Podocin expression level of USC-organoids with kECM was between USC-organoids and renal organoids. d The expression level of kidney glomerular marker Synaptopodin. The Synaptopodin expression level of USC-organoids with kECM was between USC-organoids and renal organoids. The AQP1, EPO, Podocin, and Synaptopodin expression level of USCs, USC-organoids, and USC-organoids with kECM were all significantly lower than renal organoids (three replicated samples for each group, data presented as mean ± SD)

Techniques Used: Expressing, Quantitative RT-PCR, Marker

Histology of 3D organoids (USC-organoids, USC-organoids with kECM, and renal organoids). a H.E. staining. The cell nucleus was blue stained, and cytoplasm was red stained. b Immunofluorescence staining for specific proximal tubule AQP1, kidney endocrine product EPO, and kidney glomerular markers Podocin and Synaptopodin. AQP1, EPO, and Podocin and Synaptopodin were labeled as red and DAPI as blue. The fluorescence intensity was quantified. c Whole mount staining for AQP1 and EPO. Red fluorescence indicated AQP1 and EPO and DAPI as blue. The whole mount staining provided 3D vision. The fluorescence intensity was quantified (scale bar 200 μm)
Figure Legend Snippet: Histology of 3D organoids (USC-organoids, USC-organoids with kECM, and renal organoids). a H.E. staining. The cell nucleus was blue stained, and cytoplasm was red stained. b Immunofluorescence staining for specific proximal tubule AQP1, kidney endocrine product EPO, and kidney glomerular markers Podocin and Synaptopodin. AQP1, EPO, and Podocin and Synaptopodin were labeled as red and DAPI as blue. The fluorescence intensity was quantified. c Whole mount staining for AQP1 and EPO. Red fluorescence indicated AQP1 and EPO and DAPI as blue. The whole mount staining provided 3D vision. The fluorescence intensity was quantified (scale bar 200 μm)

Techniques Used: Staining, Immunofluorescence, Labeling, Fluorescence

15) Product Images from "The Accumulation of VEGFA in the Glomerular Basement Membrane and Its Relationship with Podocyte Injury and Proteinuria in Alport Syndrome"

Article Title: The Accumulation of VEGFA in the Glomerular Basement Membrane and Its Relationship with Podocyte Injury and Proteinuria in Alport Syndrome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0135648

The expression of synaptopodin, nephrin and p-nephrin in glomeruli (immunofluorescence, a, e and i: control patients; b, f and j: AS patients with mild proteinuria; and c, g and k: AS patients with heavy proteinuria). The expression of synaptopodin and nephrin were apparent and regular in both control patients (a and e) and AS patients with mild proteinuria (b and f), but it was prominently down-regulated in AS patients with heavy proteinuria (c and g). The level of p-nephrin was low or undetectable in control patients and in AS patients with mild proteinruia (i and j), and it was significantly up-regulated in AS patients with heavy proteinuria (k). d, h and l were comparison analyses of the immunofluorescence optical density of synaptopodin, nephrin and p-nephrin for the 3 groups (Group 1: control patients, group 2: AS patients with mild and moderate proteinuria, and group 3: AS patients with heavy proteinuria. Magnifications: a-c, e-g and i-k were 400, and the scar bar in 1a is 50μm and applicable to all of the 9 immunofluorescence Figures. *, p
Figure Legend Snippet: The expression of synaptopodin, nephrin and p-nephrin in glomeruli (immunofluorescence, a, e and i: control patients; b, f and j: AS patients with mild proteinuria; and c, g and k: AS patients with heavy proteinuria). The expression of synaptopodin and nephrin were apparent and regular in both control patients (a and e) and AS patients with mild proteinuria (b and f), but it was prominently down-regulated in AS patients with heavy proteinuria (c and g). The level of p-nephrin was low or undetectable in control patients and in AS patients with mild proteinruia (i and j), and it was significantly up-regulated in AS patients with heavy proteinuria (k). d, h and l were comparison analyses of the immunofluorescence optical density of synaptopodin, nephrin and p-nephrin for the 3 groups (Group 1: control patients, group 2: AS patients with mild and moderate proteinuria, and group 3: AS patients with heavy proteinuria. Magnifications: a-c, e-g and i-k were 400, and the scar bar in 1a is 50μm and applicable to all of the 9 immunofluorescence Figures. *, p

Techniques Used: Expressing, Immunofluorescence

16) Product Images from "3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening"

Article Title: 3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening

Journal: Nature Communications

doi: 10.1038/s41467-018-07594-z

Primary podocytes can be cultured from kidney organoid glomeruli. a Isolated organoid glomeruli show evidence of podocyte cell migration (OrgPods) displaying thin arborized projections (TAPs) (inset). Inverted image shown to provide maximum contrast, scale bar 100 µm. b TAPS from newly emerged podocytes are composed of F-actin shown by phallodin immunofluorescent staining. Inverted image, scale bar 50 µm. c Immunostaining at 36 h post-plating shows a strong positively stained 3D OrgGlom with a migrating 2D OrgPod population. Left panel 2D images, right panel 3D reconstruction of Z-stack. Scale bars 50 µm. d At 48 h post-plating OrgPods display a flattened, arborized morphology with processes connecting adjacent cells (arrow), scale bar 50 µm. e Immunostaining of ciPods for SYNAPTOPODIN showed expression is absent in undifferentiated cells (ciPod: Un), only becoming evident following 14 days induced differentiation at 37 °C (ciPod: Diff). OrgPods also display strong SYNAPTOPODIN protein expression, aligned with F-actin stress fibres. Scale bars 100 µm. f OrgPods express the neonatal Fc receptor (FcRN) and actively endocytose fluorescein isothiocyanate (FITC)-labelled albumin at 37 °C resulting in FITC-accumulation in endosomes on the cell surface. This is process halted when performed at 4 °C. Scale bars 50 µm. g OrgPods stimulated with insulin (10 mg/ml) for 10 min showed cortical reorganisation of their actin cytoskeleton with GLUT4 translocation from a vesicular to plasma membrane localisation. Scale bars 50 µm. All representative images reflect a minimum of three biological replicates. For immunofluorescence, images are shown in greyscale for single channels, and merged images in colour
Figure Legend Snippet: Primary podocytes can be cultured from kidney organoid glomeruli. a Isolated organoid glomeruli show evidence of podocyte cell migration (OrgPods) displaying thin arborized projections (TAPs) (inset). Inverted image shown to provide maximum contrast, scale bar 100 µm. b TAPS from newly emerged podocytes are composed of F-actin shown by phallodin immunofluorescent staining. Inverted image, scale bar 50 µm. c Immunostaining at 36 h post-plating shows a strong positively stained 3D OrgGlom with a migrating 2D OrgPod population. Left panel 2D images, right panel 3D reconstruction of Z-stack. Scale bars 50 µm. d At 48 h post-plating OrgPods display a flattened, arborized morphology with processes connecting adjacent cells (arrow), scale bar 50 µm. e Immunostaining of ciPods for SYNAPTOPODIN showed expression is absent in undifferentiated cells (ciPod: Un), only becoming evident following 14 days induced differentiation at 37 °C (ciPod: Diff). OrgPods also display strong SYNAPTOPODIN protein expression, aligned with F-actin stress fibres. Scale bars 100 µm. f OrgPods express the neonatal Fc receptor (FcRN) and actively endocytose fluorescein isothiocyanate (FITC)-labelled albumin at 37 °C resulting in FITC-accumulation in endosomes on the cell surface. This is process halted when performed at 4 °C. Scale bars 50 µm. g OrgPods stimulated with insulin (10 mg/ml) for 10 min showed cortical reorganisation of their actin cytoskeleton with GLUT4 translocation from a vesicular to plasma membrane localisation. Scale bars 50 µm. All representative images reflect a minimum of three biological replicates. For immunofluorescence, images are shown in greyscale for single channels, and merged images in colour

Techniques Used: Cell Culture, Isolation, Migration, Staining, Immunostaining, Expressing, Translocation Assay, Immunofluorescence

17) Product Images from "Urine synaptopodin excretion is an important marker of glomerular disease progression"

Article Title: Urine synaptopodin excretion is an important marker of glomerular disease progression

Journal: The Korean Journal of Internal Medicine

doi: 10.3904/kjim.2015.226

Urine synaptopodin excretion showed significant statistical correlation with serum creatinine (SCr) in all the patients with glomerulopathy.
Figure Legend Snippet: Urine synaptopodin excretion showed significant statistical correlation with serum creatinine (SCr) in all the patients with glomerulopathy.

Techniques Used:

Amount of urine synaptopodin is significantly increased in the patients with decreased estimated glomerular filtration rate (eGFR) by Kruskal-Wallis rank sum test ( p = 0.0006).
Figure Legend Snippet: Amount of urine synaptopodin is significantly increased in the patients with decreased estimated glomerular filtration rate (eGFR) by Kruskal-Wallis rank sum test ( p = 0.0006).

Techniques Used: Filtration

18) Product Images from "Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *"

Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.593830

Delayed lithium therapy potentiates glomerular adaptation to podocyte depletion in adriamycin nephropathy. A, fluorescence immunohistochemistry staining of kidney specimens procured on day 14 for synaptopodin and WT-1. Lithium chloride therapy abrogated
Figure Legend Snippet: Delayed lithium therapy potentiates glomerular adaptation to podocyte depletion in adriamycin nephropathy. A, fluorescence immunohistochemistry staining of kidney specimens procured on day 14 for synaptopodin and WT-1. Lithium chloride therapy abrogated

Techniques Used: Fluorescence, Immunohistochemistry, Staining

19) Product Images from "Human podocytes perform polarized, caveolae-dependent albumin endocytosis"

Article Title: Human podocytes perform polarized, caveolae-dependent albumin endocytosis

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00532.2013

Cultured human urine-derived podocyte-like epithelial cells (HUPEC) cells express synaptopodin. A : Western blotting of rat renal cortex proteins shows the migration position of synaptopodin. Western blotting of cultured HUPEC cells shows these cells express
Figure Legend Snippet: Cultured human urine-derived podocyte-like epithelial cells (HUPEC) cells express synaptopodin. A : Western blotting of rat renal cortex proteins shows the migration position of synaptopodin. Western blotting of cultured HUPEC cells shows these cells express

Techniques Used: Cell Culture, Derivative Assay, Western Blot, Migration

20) Product Images from "Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture"

Article Title: Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123276

Increased urinary excretion of MMP-9 and podocyte marker proteins in Zucker diabetic rats. (A) Western blot analysis of renal cortical MMP-9 protein in 20-week-old Zucker lean controls and diabetic rats. (B) Representative Western blot images show a significant increase in urinary MMP-9 and podocyte marker proteins, nephrin and synaptopodin, in association with albuminuria in 20-week-old Zucker diabetic rats compared to normal controls. (C) Gelatin zymography analysis confirms a massive increase in urinary MMP-9 and MMP-2 activities in the diabetic rats.
Figure Legend Snippet: Increased urinary excretion of MMP-9 and podocyte marker proteins in Zucker diabetic rats. (A) Western blot analysis of renal cortical MMP-9 protein in 20-week-old Zucker lean controls and diabetic rats. (B) Representative Western blot images show a significant increase in urinary MMP-9 and podocyte marker proteins, nephrin and synaptopodin, in association with albuminuria in 20-week-old Zucker diabetic rats compared to normal controls. (C) Gelatin zymography analysis confirms a massive increase in urinary MMP-9 and MMP-2 activities in the diabetic rats.

Techniques Used: Marker, Western Blot, Zymography

Expression and localization of MMP-9 in glomerular PECs. Dual labeling using antibodies specific for MMP-9 (green) and α–tubulin (red) reveals a vesicular staining pattern that MMP-9 localizes on most microtubules (A) in rat glomerular PECs. Western blot analysis shows that primary PECs express high-level of claudin-1 and low-level of synaptopodin (B). Taqman real-time PCR analysis shows a dose-dependent increase in MMP-9 mRNA when the PECs were incubated with rat serum albumin (RSA, 0.25–1 mg/ml) for 24 hrs (C), whereas MMP-2 mRNA level was not affected. Values are mean±SEM. An n of 4–6 epithelial cultures were treated for each condition; * P
Figure Legend Snippet: Expression and localization of MMP-9 in glomerular PECs. Dual labeling using antibodies specific for MMP-9 (green) and α–tubulin (red) reveals a vesicular staining pattern that MMP-9 localizes on most microtubules (A) in rat glomerular PECs. Western blot analysis shows that primary PECs express high-level of claudin-1 and low-level of synaptopodin (B). Taqman real-time PCR analysis shows a dose-dependent increase in MMP-9 mRNA when the PECs were incubated with rat serum albumin (RSA, 0.25–1 mg/ml) for 24 hrs (C), whereas MMP-2 mRNA level was not affected. Values are mean±SEM. An n of 4–6 epithelial cultures were treated for each condition; * P

Techniques Used: Expressing, Labeling, Staining, Western Blot, Real-time Polymerase Chain Reaction, Incubation

21) Product Images from "Impact of impaired cardiac function on the progression of chronic kidney disease---role of pharmacomodulation of valsartan"

Article Title: Impact of impaired cardiac function on the progression of chronic kidney disease---role of pharmacomodulation of valsartan

Journal: American Journal of Translational Research

doi:

Immunohistochemical (IHC) stain for assessment of P-cadherin and Immunofluorescent (IF) stain for identification of synaptopodin by day 60 after CKD induction. A-E. Illustrating the microscopic finding (400x) of IHC staining for identification of P-cadherin in renal glomerulus (gray color). F. Analytical results of P-cadherin expression, * vs. other groups with different symbols (†, ‡, §, ¶), p
Figure Legend Snippet: Immunohistochemical (IHC) stain for assessment of P-cadherin and Immunofluorescent (IF) stain for identification of synaptopodin by day 60 after CKD induction. A-E. Illustrating the microscopic finding (400x) of IHC staining for identification of P-cadherin in renal glomerulus (gray color). F. Analytical results of P-cadherin expression, * vs. other groups with different symbols (†, ‡, §, ¶), p

Techniques Used: Immunohistochemistry, Staining, Expressing

22) Product Images from "Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury"

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2016091021

Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2 Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α -actinin4, and nephrin as well as mesenchymal proteins desmin and α -SMA. Representative micrographs are shown. Scale bar, 10 μ m. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α -actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2 Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μ m (C) or 10 μ m (D). Quantification data are shown in the right panel. *** P
Figure Legend Snippet: Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2 Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α -actinin4, and nephrin as well as mesenchymal proteins desmin and α -SMA. Representative micrographs are shown. Scale bar, 10 μ m. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α -actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2 Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μ m (C) or 10 μ m (D). Quantification data are shown in the right panel. *** P

Techniques Used: Mouse Assay, Staining, Knock-Out, Expressing, Immunofluorescence, Isolation

23) Product Images from "Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)"

Article Title: Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Journal: Genes & Diseases

doi: 10.1016/j.gendis.2018.04.003

The imPOD cells express podocyte markers . The imPOD and parental tsPC (at 33 °C + 100U/ml γ-interferon) cells were seeded at subconfluence and stained with antibodies against the podocyte specific marker (WT-1) and Synaptopodin ( A ), as well as the slit diaphragm complex related markers (Nephrin, Tubulin and Vinculin) ( B ). Stains without primary antibodies were used as negative controls. Cell nuclei were counter-stained with DAPI. Representative images are shown.
Figure Legend Snippet: The imPOD cells express podocyte markers . The imPOD and parental tsPC (at 33 °C + 100U/ml γ-interferon) cells were seeded at subconfluence and stained with antibodies against the podocyte specific marker (WT-1) and Synaptopodin ( A ), as well as the slit diaphragm complex related markers (Nephrin, Tubulin and Vinculin) ( B ). Stains without primary antibodies were used as negative controls. Cell nuclei were counter-stained with DAPI. Representative images are shown.

Techniques Used: Staining, Marker

24) Product Images from "Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy"

Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

Journal: BMC Nephrology

doi: 10.1186/s12882-016-0390-8

Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P
Figure Legend Snippet: Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P

Techniques Used: shRNA, Expressing, Cell Culture, Derivative Assay, Chromatin Immunoprecipitation

Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P
Figure Legend Snippet: Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P

Techniques Used: Expressing, Electron Microscopy

Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P
Figure Legend Snippet: Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P

Techniques Used: shRNA, Expressing, Immunohistochemistry, Electron Microscopy

Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation
Figure Legend Snippet: Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation

Techniques Used: Expressing

25) Product Images from "Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition"

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.07.025

Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n = 4. ∗∗∗ P
Figure Legend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n = 4. ∗∗∗ P

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P
Figure Legend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence

Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n = 6 controls and 8 patients with HIVAN. ∗∗∗ P
Figure Legend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n = 6 controls and 8 patients with HIVAN. ∗∗∗ P

Techniques Used: Expressing, Labeling, Fluorescence, Software

The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P
Figure Legend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

26) Product Images from "CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis"

Article Title: CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.12579

Activated (ED1 + ) macrophages accumulate in the glomerulus and peri-glomerular areas of Wistar rats [Wistar-puromycin aminonucleoside (PAN)], but not of nude rats (nude-PAN) or macrophage-depleted Wistar rats (Wistar-PAN-CL), during the late stage of acute PAN nephrosis. (a) Representative micrographs of glomeruli labelled with antibodies directed against ED1 (green), synaptopodin (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) from Wistar rats injected with vehicle alone (control) or PAN (Wistar-PAN), or from nude rats injected with PAN (nude-PAN). The arrows show positions of glomerular and peri-glomerular activated (ED1 + ) macrophages. Results in studies of macrophage-depleted Wistar rats were similar to those shown for nude rats (not shown). Quantitative results of immunofluorescence analyses are shown in plots of intra- and peri-glomerular macrophage counts from these animals (b), including macrophage-depleted Wistar rats (Wistar-PAN-CL). Results are expressed as means ± standard error (s.e.), n = 4/group; ** P
Figure Legend Snippet: Activated (ED1 + ) macrophages accumulate in the glomerulus and peri-glomerular areas of Wistar rats [Wistar-puromycin aminonucleoside (PAN)], but not of nude rats (nude-PAN) or macrophage-depleted Wistar rats (Wistar-PAN-CL), during the late stage of acute PAN nephrosis. (a) Representative micrographs of glomeruli labelled with antibodies directed against ED1 (green), synaptopodin (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) from Wistar rats injected with vehicle alone (control) or PAN (Wistar-PAN), or from nude rats injected with PAN (nude-PAN). The arrows show positions of glomerular and peri-glomerular activated (ED1 + ) macrophages. Results in studies of macrophage-depleted Wistar rats were similar to those shown for nude rats (not shown). Quantitative results of immunofluorescence analyses are shown in plots of intra- and peri-glomerular macrophage counts from these animals (b), including macrophage-depleted Wistar rats (Wistar-PAN-CL). Results are expressed as means ± standard error (s.e.), n = 4/group; ** P

Techniques Used: Injection, Immunofluorescence

27) Product Images from "Triptolide potentiates the cytoskeleton-stabilizing activity of cyclosporine A in glomerular podocytes via a GSK3β dependent mechanism"

Article Title: Triptolide potentiates the cytoskeleton-stabilizing activity of cyclosporine A in glomerular podocytes via a GSK3β dependent mechanism

Journal: American Journal of Translational Research

doi:

). (B) After transfection, cells were treated with cyclosporine A (CsA, 0.2 µg/ml) in the presence or absence of triptolide (TPL 0.5 ). (C) Immunoblots were subjected to densitometric analysis and arbitrary units were expressed as immunoblot densitometric ratios of synaptopodin to GAPDH as folds of the HG alone treatment group (ns, not significant). (D) After the above treatments, podocytes were processed for fluorescent labeling of F-actin with rhodamine-conjugated phalloidin and counterstained with DAPI. Representative fluorescent microscopic images show changes in F-actin cytoskeleton. Bar = 25 µm. (E and F) Podocytes were lysed and subjected to the F-Actin/G-Actin in vivo ) and subjected to densitometric analysis (F). The F-actin to G-actin ratio was calculated as immunoblot densitometric ratios of F-actin to G-actin as folds of the HG alone treatment group ( n = 3; ns, not significant).
Figure Legend Snippet: ). (B) After transfection, cells were treated with cyclosporine A (CsA, 0.2 µg/ml) in the presence or absence of triptolide (TPL 0.5 ). (C) Immunoblots were subjected to densitometric analysis and arbitrary units were expressed as immunoblot densitometric ratios of synaptopodin to GAPDH as folds of the HG alone treatment group (ns, not significant). (D) After the above treatments, podocytes were processed for fluorescent labeling of F-actin with rhodamine-conjugated phalloidin and counterstained with DAPI. Representative fluorescent microscopic images show changes in F-actin cytoskeleton. Bar = 25 µm. (E and F) Podocytes were lysed and subjected to the F-Actin/G-Actin in vivo ) and subjected to densitometric analysis (F). The F-actin to G-actin ratio was calculated as immunoblot densitometric ratios of F-actin to G-actin as folds of the HG alone treatment group ( n = 3; ns, not significant).

Techniques Used: Transfection, Western Blot, Labeling, In Vivo

). (C) Differentiated podocytes were treated with low-dose cyclosporine A (CsA, 0.2 µg/ml) in the presence or absence of low-dose triptolide (TPL 0.5 ). (B and D) Immunoblots were subjected to densitometric analysis and arbitrary units were expressed respectively as immunoblot densitometric ratios of synaptopodin to GAPDH as folds of the control group. * P
Figure Legend Snippet: ). (C) Differentiated podocytes were treated with low-dose cyclosporine A (CsA, 0.2 µg/ml) in the presence or absence of low-dose triptolide (TPL 0.5 ). (B and D) Immunoblots were subjected to densitometric analysis and arbitrary units were expressed respectively as immunoblot densitometric ratios of synaptopodin to GAPDH as folds of the control group. * P

Techniques Used: Western Blot

28) Product Images from "Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition"

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.07.025

Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n = 4. ∗∗∗ P
Figure Legend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n = 4. ∗∗∗ P

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P
Figure Legend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence

Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n = 6 controls and 8 patients with HIVAN. ∗∗∗ P
Figure Legend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n = 6 controls and 8 patients with HIVAN. ∗∗∗ P

Techniques Used: Expressing, Labeling, Fluorescence, Software

The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P
Figure Legend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

29) Product Images from "Inducible pluripotent stem cell-derived mesenchymal stem cell therapy effectively protected kidney from acute ischemia-reperfusion injury"

Article Title: Inducible pluripotent stem cell-derived mesenchymal stem cell therapy effectively protected kidney from acute ischemia-reperfusion injury

Journal: American Journal of Translational Research

doi:

Identification of the expressions of ZO-1 and synaptopodin in glomeruli at day 5 after IR procedure. A-D. Immunofluorescent (IF) microscopic finding (400 ×) for identification of the expression of positively-stained ZO-1 in glomeruli (green color). E. Analytical results of ZO-1 expression, * vs. other groups with different symbols (*, †, ‡), P
Figure Legend Snippet: Identification of the expressions of ZO-1 and synaptopodin in glomeruli at day 5 after IR procedure. A-D. Immunofluorescent (IF) microscopic finding (400 ×) for identification of the expression of positively-stained ZO-1 in glomeruli (green color). E. Analytical results of ZO-1 expression, * vs. other groups with different symbols (*, †, ‡), P

Techniques Used: Expressing, Staining

30) Product Images from "Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *"

Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.593830

Delayed lithium therapy potentiates glomerular adaptation to podocyte depletion in adriamycin nephropathy. A, fluorescence immunohistochemistry staining of kidney specimens procured on day 14 for synaptopodin and WT-1. Lithium chloride therapy abrogated
Figure Legend Snippet: Delayed lithium therapy potentiates glomerular adaptation to podocyte depletion in adriamycin nephropathy. A, fluorescence immunohistochemistry staining of kidney specimens procured on day 14 for synaptopodin and WT-1. Lithium chloride therapy abrogated

Techniques Used: Fluorescence, Immunohistochemistry, Staining

31) Product Images from "Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition"

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.07.025

Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n = 4. ∗∗∗ P
Figure Legend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n = 4. ∗∗∗ P

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P
Figure Legend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence

Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n = 6 controls and 8 patients with HIVAN. ∗∗∗ P
Figure Legend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n = 6 controls and 8 patients with HIVAN. ∗∗∗ P

Techniques Used: Expressing, Labeling, Fluorescence, Software

The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P
Figure Legend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

32) Product Images from "Myosin-1c promotes E-cadherin tension and force-dependent recruitment of α-actinin to the epithelial cell junction"

Article Title: Myosin-1c promotes E-cadherin tension and force-dependent recruitment of α-actinin to the epithelial cell junction

Journal: Journal of Cell Science

doi: 10.1242/jcs.211334

Myosin-1c colocalizes with F-actin and a tension-sensitive α-actinin-4 protein on the lateral junction in polarized epithelial monolayer. (A) OS-SIM showing immunofluorescence of myosin-1c (M1c), synaptopodin (Syp, synpo), and α-actinin-4 (A4) at the apical ( z =0 µm), the sub-apical ( z =0.4 µm) and the lateral ( z =2.2 µm) junctions. For each z -image, the top panel shows the x-z , y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of six independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (B) Merge images of the representative linear junction shown in A. The Pearson's correlation coefficients between myoin-1c, synaptopodin or α-actinin-4 at the junctional vertices (white circle) and linear junctions (white lozenge) are represented by Rv and Rj, respectively. Correlations were calculated from 500–1500 pixels per protein per z -image in one set of data. Six sets of data were analyzed and a representative set of data is shown. Scale bar: 2 µm. (C) OS-SIM showing immunofluorescence of myosin-1c, actin (phalloidin, Phal), and myosin IIB (MIIB) at the sub-apical ( z =1 µm) and the lateral ( z =2.6 and 4.2 µm) junctions. For each z -image, the top panel shows the x-z, y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of three independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (D) Merge images of the representative linear junction shown in C. The Pearson's correlation coefficient ( R ) between two proteins at the linear junction was calculated from 1000–2000 pixels per protein per z -image for one set of data. Data set is representative of six sets of data from one experiment out of three independent experiments. Scale bar: 2 µm.
Figure Legend Snippet: Myosin-1c colocalizes with F-actin and a tension-sensitive α-actinin-4 protein on the lateral junction in polarized epithelial monolayer. (A) OS-SIM showing immunofluorescence of myosin-1c (M1c), synaptopodin (Syp, synpo), and α-actinin-4 (A4) at the apical ( z =0 µm), the sub-apical ( z =0.4 µm) and the lateral ( z =2.2 µm) junctions. For each z -image, the top panel shows the x-z , y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of six independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (B) Merge images of the representative linear junction shown in A. The Pearson's correlation coefficients between myoin-1c, synaptopodin or α-actinin-4 at the junctional vertices (white circle) and linear junctions (white lozenge) are represented by Rv and Rj, respectively. Correlations were calculated from 500–1500 pixels per protein per z -image in one set of data. Six sets of data were analyzed and a representative set of data is shown. Scale bar: 2 µm. (C) OS-SIM showing immunofluorescence of myosin-1c, actin (phalloidin, Phal), and myosin IIB (MIIB) at the sub-apical ( z =1 µm) and the lateral ( z =2.6 and 4.2 µm) junctions. For each z -image, the top panel shows the x-z, y-z and x-y images, and the bottom panel shows a representative linear junction from the x-y image. Data are representative of ten sets of images from one experiment out of three independent experiments. Scale bars: 10 µm (orange); 2 µm (white). (D) Merge images of the representative linear junction shown in C. The Pearson's correlation coefficient ( R ) between two proteins at the linear junction was calculated from 1000–2000 pixels per protein per z -image for one set of data. Data set is representative of six sets of data from one experiment out of three independent experiments. Scale bar: 2 µm.

Techniques Used: Immunofluorescence

Myosin-1c knockdown abolishes junction recruitment of α-actinin-4 and weakens cell-cell cohesion. (A) OS-SIM images of myosin-1c (Myo1c), α-catenin, p120-catenin and E-cadherin at the apical plane of heterogeneous myosin-1c knockdown (KD) cell monolayers. Data are representative of 12 sets of images from one experiment out of three independent experiments. (B) OS-SIM images of myosin-1c, β-catenin, synaptopodin (synpo) and α-actinin-4 at the apical plane of heterogeneous myosin-1c knockdown cell monolayers. Data are representative of 12 sets of images from one experiment out of three different independent experiments. (C) Quantification of junctional myosin-1c, α-catenin, p120-catenin, E-cadherin, β-catenin, synaptopodin and α-actinin-4 immunofluorescence in wild-type (WT) and myosin-1c knockdown (Myo1cKD) cells. Bar graphs show the normalized mean±s.e.m. intensity of 24 junctions. P
Figure Legend Snippet: Myosin-1c knockdown abolishes junction recruitment of α-actinin-4 and weakens cell-cell cohesion. (A) OS-SIM images of myosin-1c (Myo1c), α-catenin, p120-catenin and E-cadherin at the apical plane of heterogeneous myosin-1c knockdown (KD) cell monolayers. Data are representative of 12 sets of images from one experiment out of three independent experiments. (B) OS-SIM images of myosin-1c, β-catenin, synaptopodin (synpo) and α-actinin-4 at the apical plane of heterogeneous myosin-1c knockdown cell monolayers. Data are representative of 12 sets of images from one experiment out of three different independent experiments. (C) Quantification of junctional myosin-1c, α-catenin, p120-catenin, E-cadherin, β-catenin, synaptopodin and α-actinin-4 immunofluorescence in wild-type (WT) and myosin-1c knockdown (Myo1cKD) cells. Bar graphs show the normalized mean±s.e.m. intensity of 24 junctions. P

Techniques Used: Immunofluorescence

33) Product Images from "Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells"

Article Title: Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00483.2016

Distribution of NP-Cy3-siOrai1 in MCs in mouse kidney. A : representative images from 3 mice showing localization of NP-Cy3-siOrai1 (red) in glomeruli (indicated by arrows) but not in tubules. Original magnification, ×200. B : localization of NP-Cy3-siOrai1 in MCs ( top ), but not in podocytes ( bottom ), represented from 3 mice. MCs and podocytes were stained with integrin-α8 (green) and synaptopodin (green), respectively. NP-Cy3-siOrai1 was shown as red signals. Original magnification, ×200.
Figure Legend Snippet: Distribution of NP-Cy3-siOrai1 in MCs in mouse kidney. A : representative images from 3 mice showing localization of NP-Cy3-siOrai1 (red) in glomeruli (indicated by arrows) but not in tubules. Original magnification, ×200. B : localization of NP-Cy3-siOrai1 in MCs ( top ), but not in podocytes ( bottom ), represented from 3 mice. MCs and podocytes were stained with integrin-α8 (green) and synaptopodin (green), respectively. NP-Cy3-siOrai1 was shown as red signals. Original magnification, ×200.

Techniques Used: Mouse Assay, Staining

34) Product Images from "Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition"

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.07.025

Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n  = 6 controls and 8 patients with HIVAN. ∗∗∗ P ÂÂ
Figure Legend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n  = 6 controls and 8 patients with HIVAN. ∗∗∗ P ÂÂ

Techniques Used: Expressing, Labeling, Fluorescence, Software

The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n  = 4 ( A and C–F ). ∗ P ÂÂ
Figure Legend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n  = 4 ( A and C–F ). ∗ P ÂÂ

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n  = 4. ∗∗∗ P ÂÂ
Figure Legend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n  = 4. ∗∗∗ P ÂÂ

Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n  = 4 ( A and B ); n  = 3 ( E and F ). ∗ P ÂÂ
Figure Legend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n  = 4 ( A and B ); n  = 3 ( E and F ). ∗ P ÂÂ

Techniques Used: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence

35) Product Images from "Extract of Rhizoma Polygonum cuspidatum reduces early renal podocyte injury in streptozotocin-induced diabetic rats and its active compound emodin inhibits methylglyoxal-mediated glycation of proteins"

Article Title: Extract of Rhizoma Polygonum cuspidatum reduces early renal podocyte injury in streptozotocin-induced diabetic rats and its active compound emodin inhibits methylglyoxal-mediated glycation of proteins

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2015.4214

Effect of PCE on podocyte loss. Representative histology sections from the NOR, DM, PCE-100 and PCE-350 groups (magnification, ×400): (A) Synaptopodin and (B) WT-1 staining of the glomeruli. Morphometric analysis of (C) synaptopodin and (D) WT-1 in the renal cortex of rats from each group. All data are expressed as the mean ± standard error of the mean (n=8). * P
Figure Legend Snippet: Effect of PCE on podocyte loss. Representative histology sections from the NOR, DM, PCE-100 and PCE-350 groups (magnification, ×400): (A) Synaptopodin and (B) WT-1 staining of the glomeruli. Morphometric analysis of (C) synaptopodin and (D) WT-1 in the renal cortex of rats from each group. All data are expressed as the mean ± standard error of the mean (n=8). * P

Techniques Used: Staining

Effect of PCE on the apoptosis of podocytes. (A) Dual-labeling for TUNEL (brown) and synaptopodin (red) in kidney sections from the NOR, DM, PCE-100 and PCE-350 groups (magnification, ×400). Morphometric analysis of (B) the positive area of synaptopodin expression and (C) TUNEL and synaptopodin double-positive cells. Hematoxylin (blue) was used to counterstain the nucleus in (A). All data are expressed as the mean ± standard error of the mean (n=8). * P
Figure Legend Snippet: Effect of PCE on the apoptosis of podocytes. (A) Dual-labeling for TUNEL (brown) and synaptopodin (red) in kidney sections from the NOR, DM, PCE-100 and PCE-350 groups (magnification, ×400). Morphometric analysis of (B) the positive area of synaptopodin expression and (C) TUNEL and synaptopodin double-positive cells. Hematoxylin (blue) was used to counterstain the nucleus in (A). All data are expressed as the mean ± standard error of the mean (n=8). * P

Techniques Used: Labeling, TUNEL Assay, Expressing

36) Product Images from "Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury"

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2016091021

Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2 Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α -actinin4, and nephrin as well as mesenchymal proteins desmin and α -SMA. Representative micrographs are shown. Scale bar, 10 μ m. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α -actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2 Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μ m (C) or 10 μ m (D). Quantification data are shown in the right panel. *** P
Figure Legend Snippet: Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2 Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α -actinin4, and nephrin as well as mesenchymal proteins desmin and α -SMA. Representative micrographs are shown. Scale bar, 10 μ m. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α -actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2 Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μ m (C) or 10 μ m (D). Quantification data are shown in the right panel. *** P

Techniques Used: Mouse Assay, Staining, Knock-Out, Expressing, Immunofluorescence, Isolation

37) Product Images from "The AGE receptor, OST48 drives podocyte foot process effacement and basement membrane expansion in experimental diabetic kidney disease via promotion of endoplasmic reticulum stress"

Article Title: The AGE receptor, OST48 drives podocyte foot process effacement and basement membrane expansion in experimental diabetic kidney disease via promotion of endoplasmic reticulum stress

Journal: bioRxiv

doi: 10.1101/710186

Podocyte OST48 increased ER stress markers. ( A ) Confocal photomicrographs of either ER stress marker GRP-78 (green), or XBP-1 (green) and podocyte foot process marker, synaptopodin (red). ( B - C ) Heat map representation of SWATH-MS proteomics data for enzymatic pathways involved in ( B ) endoplasmic reticulum (ER) stress and oxidative stress (OS) enzymatic pathways, and ( C ) ubiquinone biosynthesis pathways. Significant proteins are represented as bolded cells, where red indicates an increase and blue indicates a decrease in protein concentrations. Data represented as means ± SD (n = 4-7/group). Scale bars from representative images of confocal microscopy were 30µm. Data represented as means ± SD (n = 5/group). For proteomics, MSstatsV2.6.4 determined significant (P
Figure Legend Snippet: Podocyte OST48 increased ER stress markers. ( A ) Confocal photomicrographs of either ER stress marker GRP-78 (green), or XBP-1 (green) and podocyte foot process marker, synaptopodin (red). ( B - C ) Heat map representation of SWATH-MS proteomics data for enzymatic pathways involved in ( B ) endoplasmic reticulum (ER) stress and oxidative stress (OS) enzymatic pathways, and ( C ) ubiquinone biosynthesis pathways. Significant proteins are represented as bolded cells, where red indicates an increase and blue indicates a decrease in protein concentrations. Data represented as means ± SD (n = 4-7/group). Scale bars from representative images of confocal microscopy were 30µm. Data represented as means ± SD (n = 5/group). For proteomics, MSstatsV2.6.4 determined significant (P

Techniques Used: Marker, Confocal Microscopy

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Santa Cruz Biotechnology synaptopodin
    Effects of conditioned MSCs on expression of <t>synaptopodin</t> and fibronectin in podocytes. Podocytes were incubated without or with 15 ng/ml TGF-β1, conditioned MSCs and/or ascorbic acid for 72 hrs. Cell lysates were analysed with Western blotting. ( A ) Representative Western blot and bar graph analysis of relative protein level of synaptopodin, which were normalized to control. ( B ) Bar graph analysis of quantitative PCR analysis of relative fibronectin expression to GAPDH normalized to control. * P
    Synaptopodin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synaptopodin/product/Santa Cruz Biotechnology
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synaptopodin - by Bioz Stars, 2022-05
    85/100 stars
      Buy from Supplier

    86
    Santa Cruz Biotechnology synaptopodin antibody
    SQ alleviated glomerular podocyte injury in PHN rats. Effects of SQ and CP on foot process width (magnification × 12,000, red arrows) and <t>synaptopodin</t> expression (magnification × 400) were measured by TEM and immunofluorescence staining (A) . With the treatment of SQ and CP, restored glomerular podocytic foot processes (B) and synaptopodin expression (C) were seen in PHN rats ( n = 6). Data are represented as mean ± SD from independent groups. ** p
    Synaptopodin Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synaptopodin antibody/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synaptopodin antibody - by Bioz Stars, 2022-05
    86/100 stars
      Buy from Supplier

    Image Search Results


    Effects of conditioned MSCs on expression of synaptopodin and fibronectin in podocytes. Podocytes were incubated without or with 15 ng/ml TGF-β1, conditioned MSCs and/or ascorbic acid for 72 hrs. Cell lysates were analysed with Western blotting. ( A ) Representative Western blot and bar graph analysis of relative protein level of synaptopodin, which were normalized to control. ( B ) Bar graph analysis of quantitative PCR analysis of relative fibronectin expression to GAPDH normalized to control. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation

    doi: 10.1111/j.1582-4934.2012.01610.x

    Figure Lengend Snippet: Effects of conditioned MSCs on expression of synaptopodin and fibronectin in podocytes. Podocytes were incubated without or with 15 ng/ml TGF-β1, conditioned MSCs and/or ascorbic acid for 72 hrs. Cell lysates were analysed with Western blotting. ( A ) Representative Western blot and bar graph analysis of relative protein level of synaptopodin, which were normalized to control. ( B ) Bar graph analysis of quantitative PCR analysis of relative fibronectin expression to GAPDH normalized to control. * P

    Article Snippet: The membrane was incubated with 1A4 anti-α- SMA (Clone1A4, 1:1000 dilution; Sigma-Aldrich), fibronectin (H-300, 1:400 dilution; Santa Cruz, CA, USA) or synaptopodin (H-140, 1:1000 dilution; Santa Cruz) mAb.

    Techniques: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction

    SQ alleviated glomerular podocyte injury in PHN rats. Effects of SQ and CP on foot process width (magnification × 12,000, red arrows) and synaptopodin expression (magnification × 400) were measured by TEM and immunofluorescence staining (A) . With the treatment of SQ and CP, restored glomerular podocytic foot processes (B) and synaptopodin expression (C) were seen in PHN rats ( n = 6). Data are represented as mean ± SD from independent groups. ** p

    Journal: Frontiers in Pharmacology

    Article Title: Sanqi Oral Solution Mitigates Proteinuria in Rat Passive Heymann Nephritis and Blocks Podocyte Apoptosis via Nrf2/HO-1 Pathway

    doi: 10.3389/fphar.2021.727874

    Figure Lengend Snippet: SQ alleviated glomerular podocyte injury in PHN rats. Effects of SQ and CP on foot process width (magnification × 12,000, red arrows) and synaptopodin expression (magnification × 400) were measured by TEM and immunofluorescence staining (A) . With the treatment of SQ and CP, restored glomerular podocytic foot processes (B) and synaptopodin expression (C) were seen in PHN rats ( n = 6). Data are represented as mean ± SD from independent groups. ** p

    Article Snippet: Primary antibodies against C5b-9 (sc-66190) and synaptopodin (sc-515842) were purchased from Santa Cruz Biotechnology (Dallas, Texas, United States).

    Techniques: Expressing, Transmission Electron Microscopy, Immunofluorescence, Staining