Structured Review

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
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1) Product Images from "Sanqi Oral Solution Mitigates Proteinuria in Rat Passive Heymann Nephritis and Blocks Podocyte Apoptosis via Nrf2/HO-1 Pathway"

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

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.727874

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
Figure Legend 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

Techniques Used: Expressing, Transmission Electron Microscopy, Immunofluorescence, Staining

2) Product Images from "Age-related GSK3 β overexpression drives podocyte senescence and glomerular aging"

Article Title: Age-related GSK3 β overexpression drives podocyte senescence and glomerular aging

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI141848

Long-term lithium carbonate therapy in psychiatric patients inhibits GSK3β activity and attenuates cellular senescence in urinary exfoliated cells. ( A ) Schematic diagram depicts preparation of urinary exfoliated cells from psychiatric patients treated either with lithium carbonate [Li (+), n = 12] or without lithium carbonate [Li (–), n = 12]. Scale bars: 100 μm. ( B ) Immunofluorescent staining of urinary exfoliated cells for synaptopodin (red) with DAPI counterstaining for nuclei, as shown by fluorescence microscopy and differential interference contrast (DIC) microscopy. Arrowheads indicate synaptopodin-positive podocytes, while arrows indicate synaptopodin-negative urinary cells. Scale bars: 20 μm. ( C ) Multicolor immunofluorescent staining of urinary exfoliated cells for phosphorylated GSK3β at serine 9 (p-GSK3β S9 ), p16 INK4A , and WT-1. Arrows indicate WT-1–positive urinary podocytes with p-GSK3β S9-lo p16 hi staining pattern. Arrowheads indicate WT-1–positive urinary podocytes with p-GSK3β S9-hi p16 lo staining pattern. Scale bars: 100 μm. ( D ) Quantification of cells with high and low expression of p16 INK4A among all WT-1 + urinary cells. ** P
Figure Legend Snippet: Long-term lithium carbonate therapy in psychiatric patients inhibits GSK3β activity and attenuates cellular senescence in urinary exfoliated cells. ( A ) Schematic diagram depicts preparation of urinary exfoliated cells from psychiatric patients treated either with lithium carbonate [Li (+), n = 12] or without lithium carbonate [Li (–), n = 12]. Scale bars: 100 μm. ( B ) Immunofluorescent staining of urinary exfoliated cells for synaptopodin (red) with DAPI counterstaining for nuclei, as shown by fluorescence microscopy and differential interference contrast (DIC) microscopy. Arrowheads indicate synaptopodin-positive podocytes, while arrows indicate synaptopodin-negative urinary cells. Scale bars: 20 μm. ( C ) Multicolor immunofluorescent staining of urinary exfoliated cells for phosphorylated GSK3β at serine 9 (p-GSK3β S9 ), p16 INK4A , and WT-1. Arrows indicate WT-1–positive urinary podocytes with p-GSK3β S9-lo p16 hi staining pattern. Arrowheads indicate WT-1–positive urinary podocytes with p-GSK3β S9-hi p16 lo staining pattern. Scale bars: 100 μm. ( D ) Quantification of cells with high and low expression of p16 INK4A among all WT-1 + urinary cells. ** P

Techniques Used: Activity Assay, Staining, Fluorescence, Microscopy, Expressing

Cellular senescence and senescence-associated secretory phenotypes (SASPs) are mitigated in primary podocytes derived from KO mice and reinstated after GSK3β reconstitution. ( A ) Primary podocytes were cultured from glomeruli isolated from 12-month-old control mice (Con) and mice with podocyte-specific GSK3β knockout (KO). Representative micrographs show freshly isolated glomeruli and primary cultures of podocytes. Scale bars: 75 μm. ( B – E ) Primary podocytes were subjected to electroporation-based transfection with either an empty plasmid vector (EV) or a plasmid encoding the HA-conjugated WT GSK3β by using the Amaxa Nucleofection kit. ( B ) Cells were processed for SA-β-gal activity staining or immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm (top 2 rows) and 30 μm (bottom row). ( C ) Cell lysates were processed for immunoblot analysis for indicated proteins, including SASP factors like fibronectin (FN) and PAI-1. β-Tubulin served as a loading control. ( D ) Absolute count of the number of γH2AX + cells expressed as percentages of the total number of cells per microscopic field. * P
Figure Legend Snippet: Cellular senescence and senescence-associated secretory phenotypes (SASPs) are mitigated in primary podocytes derived from KO mice and reinstated after GSK3β reconstitution. ( A ) Primary podocytes were cultured from glomeruli isolated from 12-month-old control mice (Con) and mice with podocyte-specific GSK3β knockout (KO). Representative micrographs show freshly isolated glomeruli and primary cultures of podocytes. Scale bars: 75 μm. ( B – E ) Primary podocytes were subjected to electroporation-based transfection with either an empty plasmid vector (EV) or a plasmid encoding the HA-conjugated WT GSK3β by using the Amaxa Nucleofection kit. ( B ) Cells were processed for SA-β-gal activity staining or immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm (top 2 rows) and 30 μm (bottom row). ( C ) Cell lysates were processed for immunoblot analysis for indicated proteins, including SASP factors like fibronectin (FN) and PAI-1. β-Tubulin served as a loading control. ( D ) Absolute count of the number of γH2AX + cells expressed as percentages of the total number of cells per microscopic field. * P

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Isolation, Knock-Out, Electroporation, Transfection, Plasmid Preparation, Activity Assay, Staining

GSK3β regulates the phosphorylation of p16 INK4A and p53, resulting in modulation of senescence signaling in podocytes. Conditionally immortalized murine podocytes were transiently lipotransfected with a control empty plasmid vector (EV), or plasmids encoding the HA-conjugated dominant-negative kinase dead (KD) mutant of GSK3β or constitutively active (S9A) mutant of GSK3β in the presence or absence of lithium chloride (LiCl, 10 mM) or an equal volume of vehicle. ( A ) After different treatments, cells were subjected to immunofluorescent staining for HA, which revealed a transfection efficiency of approximately 80%. Scale bar: 20 μm. ( B ) Whole cell lysates were processed for immunoprecipitation (IP) by using an anti-p16 INK4A or -p53 antibody, followed by immunoblot analysis (IB) of immunoprecipitates for phosphorylated serine (p-Ser), in parallel with input controls. ( C ) Representative immunoblot analysis of cell lysates for indicated molecules. β-Tubulin served as a loading control. ( D ) Cells were subjected to SA-β-gal activity staining, or to immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm. ( E ) Absolute count of the number of γH2AX + cells as percentages of the total number of cells per microscopic field. * P
Figure Legend Snippet: GSK3β regulates the phosphorylation of p16 INK4A and p53, resulting in modulation of senescence signaling in podocytes. Conditionally immortalized murine podocytes were transiently lipotransfected with a control empty plasmid vector (EV), or plasmids encoding the HA-conjugated dominant-negative kinase dead (KD) mutant of GSK3β or constitutively active (S9A) mutant of GSK3β in the presence or absence of lithium chloride (LiCl, 10 mM) or an equal volume of vehicle. ( A ) After different treatments, cells were subjected to immunofluorescent staining for HA, which revealed a transfection efficiency of approximately 80%. Scale bar: 20 μm. ( B ) Whole cell lysates were processed for immunoprecipitation (IP) by using an anti-p16 INK4A or -p53 antibody, followed by immunoblot analysis (IB) of immunoprecipitates for phosphorylated serine (p-Ser), in parallel with input controls. ( C ) Representative immunoblot analysis of cell lysates for indicated molecules. β-Tubulin served as a loading control. ( D ) Cells were subjected to SA-β-gal activity staining, or to immunofluorescent staining for synaptopodin (SYNPO; red) or γH2AX (green) followed by counterstaining with DAPI for nuclei or with rhodamine-phalloidin for F-actin (red). Scale bars: 20 μm. ( E ) Absolute count of the number of γH2AX + cells as percentages of the total number of cells per microscopic field. * P

Techniques Used: Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Staining, Transfection, Immunoprecipitation, Activity Assay

3) Product Images from "Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction"

Article Title: Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction

Journal: bioRxiv

doi: 10.1101/2020.12.30.424702

Synaptopodin is responsible for 2 new actomyosin structures at the apical junction to support extrinsic and intrinsic contractility. (A) Comparison of actomyosin meshwork and cables with apical stress fiber, type I, and contractomere, type II, actomyosin organizations. Apical stress fibers can selectively link neighboring junctions as well as junctions from opposite sides of a cell. Contractomere is a unique actomyosin structure that contains non-filamentous myosin II. (B) Comparison between actomyosin meshwork and apical stress fibers is shown at the top panels. Apical stress fibers inserted at cell-cell adhesion can propagate force and generate tissue-level tension over many cells in an epithelial monolayer, the bottom panel. (C) Shortening and lengthening the junction by “walking” the contractomere. Motility of contractomeres contributes to apical junction constriction during cell extrusion and pure-string wound closure. The prevailing model of junction remodeling requires disassembly of the actomyosin cortex and endocytosis of existing junction to shorten a junction. (D) Contractomere is directly powered by myosin II and actin polymerization to generate force locally at the junction while apical stress fiber powered by myosin II are at a distance away from the junction. Contractomeres generate intrinsic force whereas apical stress fibers or actomyosin meshworks generate extrinsic force with respect to the junction. (E) Key to cartoon.
Figure Legend Snippet: Synaptopodin is responsible for 2 new actomyosin structures at the apical junction to support extrinsic and intrinsic contractility. (A) Comparison of actomyosin meshwork and cables with apical stress fiber, type I, and contractomere, type II, actomyosin organizations. Apical stress fibers can selectively link neighboring junctions as well as junctions from opposite sides of a cell. Contractomere is a unique actomyosin structure that contains non-filamentous myosin II. (B) Comparison between actomyosin meshwork and apical stress fibers is shown at the top panels. Apical stress fibers inserted at cell-cell adhesion can propagate force and generate tissue-level tension over many cells in an epithelial monolayer, the bottom panel. (C) Shortening and lengthening the junction by “walking” the contractomere. Motility of contractomeres contributes to apical junction constriction during cell extrusion and pure-string wound closure. The prevailing model of junction remodeling requires disassembly of the actomyosin cortex and endocytosis of existing junction to shorten a junction. (D) Contractomere is directly powered by myosin II and actin polymerization to generate force locally at the junction while apical stress fiber powered by myosin II are at a distance away from the junction. Contractomeres generate intrinsic force whereas apical stress fibers or actomyosin meshworks generate extrinsic force with respect to the junction. (E) Key to cartoon.

Techniques Used:

Myosin IIB is absent from the apical junction in synaptopodin knockdown cells despite the presence of actin. Synaptopodin knockdowns electively affects myosin IIB but not actin in mature MDCK monolayer.
Figure Legend Snippet: Myosin IIB is absent from the apical junction in synaptopodin knockdown cells despite the presence of actin. Synaptopodin knockdowns electively affects myosin IIB but not actin in mature MDCK monolayer.

Techniques Used:

Superresolution immunofluorescence microscopy showing the assembly and disassembly of 2 actomyosin structures during junction maturation. (A) Apical stress fiber, corresponding to type I actomyosin organization shown in the left panel and the graph, has alternating myosin IIB and synaptopodin pattern and lies parallel to E-cadherin junction. Orange arrowheads point to the end of an apical stress fiber marked by E-cadherin. White arrowheads point to the junctional region where synaptopodin is in close proximity to E-cadherin. White long arrow shows the junctional length used for x-axis of the graph. (B) Apical stress fiber is inserted head-on at E-cadherin junction, marked by orange arrowhead. Synaptopodin overlaps with E-cadherin at the insertion point of apical stress fiber, in white circle. White arrowheads mark sites where synaptopodin is in close proximity to or overlapping with E-cadherin. (C) Disassembly of apical stress fibers in maturing junction and loss of alternating pattern of synaptopodin and myosin IIB as shown in graph. The disassembly of apical stress fiber coincides with the formation of type II actomyosin structures containing myosin IIB, synaptopodin, and alpha-actinin-4, as shown in graph. White circles in the left panels show type II structures. White arrowheads mark the colocalization of myosin IIB, synaptopodin, alpha-actinin-4. White long arrow shows the junctional length used for x-axis of the graph. (D) Apical stress fibers disappear upon junction maturation whereas type II actomyosin structures are prominent at mature apical junctions. Left panel are X-Y, Y-Z, and X-Z views of mature polarized MDCK monolayers. Small white boxed areas are enlarged to show colocalization of myosin IIB, synaptopodin, and alpha-actinin-4. (E) Actin accumulates at type II actomyosin structure, circled in white, colocalizing with myosin IIB, synaptopodin, and alpha-actinin-4 marked by white arrowheads. Graph shows the absence of apical stress fiber and the presence type II structure. White long arrow shows the junctional length used for x-axis of the graph.
Figure Legend Snippet: Superresolution immunofluorescence microscopy showing the assembly and disassembly of 2 actomyosin structures during junction maturation. (A) Apical stress fiber, corresponding to type I actomyosin organization shown in the left panel and the graph, has alternating myosin IIB and synaptopodin pattern and lies parallel to E-cadherin junction. Orange arrowheads point to the end of an apical stress fiber marked by E-cadherin. White arrowheads point to the junctional region where synaptopodin is in close proximity to E-cadherin. White long arrow shows the junctional length used for x-axis of the graph. (B) Apical stress fiber is inserted head-on at E-cadherin junction, marked by orange arrowhead. Synaptopodin overlaps with E-cadherin at the insertion point of apical stress fiber, in white circle. White arrowheads mark sites where synaptopodin is in close proximity to or overlapping with E-cadherin. (C) Disassembly of apical stress fibers in maturing junction and loss of alternating pattern of synaptopodin and myosin IIB as shown in graph. The disassembly of apical stress fiber coincides with the formation of type II actomyosin structures containing myosin IIB, synaptopodin, and alpha-actinin-4, as shown in graph. White circles in the left panels show type II structures. White arrowheads mark the colocalization of myosin IIB, synaptopodin, alpha-actinin-4. White long arrow shows the junctional length used for x-axis of the graph. (D) Apical stress fibers disappear upon junction maturation whereas type II actomyosin structures are prominent at mature apical junctions. Left panel are X-Y, Y-Z, and X-Z views of mature polarized MDCK monolayers. Small white boxed areas are enlarged to show colocalization of myosin IIB, synaptopodin, and alpha-actinin-4. (E) Actin accumulates at type II actomyosin structure, circled in white, colocalizing with myosin IIB, synaptopodin, and alpha-actinin-4 marked by white arrowheads. Graph shows the absence of apical stress fiber and the presence type II structure. White long arrow shows the junctional length used for x-axis of the graph.

Techniques Used: Immunofluorescence, Microscopy

Myosin IIA is present at the apical junction in synaptopodin knockdown monolayers. (A) Junctional myosin IIA level is unchanged in synaptopodin knockdown cells. (B) Myosin IIA basal stress fibers are converted to myosin IIA meshworks in synaptopodin knockdown cells.
Figure Legend Snippet: Myosin IIA is present at the apical junction in synaptopodin knockdown monolayers. (A) Junctional myosin IIA level is unchanged in synaptopodin knockdown cells. (B) Myosin IIA basal stress fibers are converted to myosin IIA meshworks in synaptopodin knockdown cells.

Techniques Used:

Actin polymerization and myosin II motility are coupled at the contractomere. (A) Contractomeres, in circles, are marked by myosin IIB, actin, and alpha-actinin-4, an essential factor for actin polymerization at the junction. Images are immunofluorescence of mature MDCK cell monolayer. (B) Contractomeres in purified membranes, arrows, are marked by myosin IIB, synaptopodin, and alpha-actinin-4. Images are immunofluorescence of purified junction-enriched membranes. (C) Inhibition of actin assembly and membrane contraction by blebbistatin in ex vivo junctional membrane. In vitro actin assembly assays was performed on purified junction-enriched membranes using rhodamine-labelled red actin monomers in the absence or presence of blebbistatin. Frames from time-lapse movies of actin polymerization at the contractomeres are shown. (D) Contractomeric myosin IIB marks the site of actin assembly on purified membrane. Immunofluorescence for myosin IIB were performed after the actin assembly reaction on purified membrane. (E) Negative-stain electron microscopy showing actin filaments inserted at large electron-dense contractomeres. Contractomeres lack the characteristic bipolar myosin minifilaments but can be mechanically and chemically dissociated into sub-complexes, including a sub-complex containing myosin II monomer. The 2 motor heads of the myosin II monomer interact with actin filament (yellow arrows) and the tail region of the myosin II monomer interacts with electron-dense materials (red arrows). (F) Protocol for reconstitution actin assembly assay using stripped membranes and recombinant alpha-actinin-4 full-length or truncated proteins. Bottom panels shows that alpha-actinin-4 with one actin-binding domain failed to crosslink Alexa 647-labelled actin filaments into bundles that can be readily visualized under the light microscope. (G) Actin assembly on the contractomere requires interaction of alpha-actinin-4 with actin. Reconstitution actin assembly assay showing alpha-actinin-4 lacking the actin-binding domains was recruited to the contractomere but failed to support actin assembly. Graph shows intensity measurement of Oregon green-labelled alpha-actinin-4 on individual contractomeres; boxes represent 75 percentile and error bars are standard deviation. (H) Contractomeric actin assembly requires only one actin-binding domain of alpha-actinin-4. Graph shows intensity measurement of rhodamine-labelled actin on individual contractomeres. Bars mark the means.
Figure Legend Snippet: Actin polymerization and myosin II motility are coupled at the contractomere. (A) Contractomeres, in circles, are marked by myosin IIB, actin, and alpha-actinin-4, an essential factor for actin polymerization at the junction. Images are immunofluorescence of mature MDCK cell monolayer. (B) Contractomeres in purified membranes, arrows, are marked by myosin IIB, synaptopodin, and alpha-actinin-4. Images are immunofluorescence of purified junction-enriched membranes. (C) Inhibition of actin assembly and membrane contraction by blebbistatin in ex vivo junctional membrane. In vitro actin assembly assays was performed on purified junction-enriched membranes using rhodamine-labelled red actin monomers in the absence or presence of blebbistatin. Frames from time-lapse movies of actin polymerization at the contractomeres are shown. (D) Contractomeric myosin IIB marks the site of actin assembly on purified membrane. Immunofluorescence for myosin IIB were performed after the actin assembly reaction on purified membrane. (E) Negative-stain electron microscopy showing actin filaments inserted at large electron-dense contractomeres. Contractomeres lack the characteristic bipolar myosin minifilaments but can be mechanically and chemically dissociated into sub-complexes, including a sub-complex containing myosin II monomer. The 2 motor heads of the myosin II monomer interact with actin filament (yellow arrows) and the tail region of the myosin II monomer interacts with electron-dense materials (red arrows). (F) Protocol for reconstitution actin assembly assay using stripped membranes and recombinant alpha-actinin-4 full-length or truncated proteins. Bottom panels shows that alpha-actinin-4 with one actin-binding domain failed to crosslink Alexa 647-labelled actin filaments into bundles that can be readily visualized under the light microscope. (G) Actin assembly on the contractomere requires interaction of alpha-actinin-4 with actin. Reconstitution actin assembly assay showing alpha-actinin-4 lacking the actin-binding domains was recruited to the contractomere but failed to support actin assembly. Graph shows intensity measurement of Oregon green-labelled alpha-actinin-4 on individual contractomeres; boxes represent 75 percentile and error bars are standard deviation. (H) Contractomeric actin assembly requires only one actin-binding domain of alpha-actinin-4. Graph shows intensity measurement of rhodamine-labelled actin on individual contractomeres. Bars mark the means.

Techniques Used: Immunofluorescence, Purification, Inhibition, Ex Vivo, In Vitro, Staining, Electron Microscopy, Recombinant, Binding Assay, Light Microscopy, Standard Deviation

MDCK cell expresses synaptopodin isoform A. (A) Synaptopodin is encoded by 3 exons, resulting in 4 splice variants. We have raised antibodies to each spliced region to characterize the expression of synaptopodin isoforms in different mammalian tissues and cells. Table summarized results from western blot studies using our newly generated antibodies (see methods section). (B) Western blot using antibodies against region encoded by exon 2, thus recognizing all synaptopodin isoforms.
Figure Legend Snippet: MDCK cell expresses synaptopodin isoform A. (A) Synaptopodin is encoded by 3 exons, resulting in 4 splice variants. We have raised antibodies to each spliced region to characterize the expression of synaptopodin isoforms in different mammalian tissues and cells. Table summarized results from western blot studies using our newly generated antibodies (see methods section). (B) Western blot using antibodies against region encoded by exon 2, thus recognizing all synaptopodin isoforms.

Techniques Used: Expressing, Western Blot, Generated

Live-imaging of synaptopodin showing 2 apical actomyosin structures and basal stress fibers in MDCK cell monolayers. (A) Apical stress fibers are outlined by aligned and repeated synaptopodin densities, circled in blue, yellow, and green. Apical stress fibers are inserted at the junction via synaptopodin linkers, highlighted in pink, using 2 configurations, either head-on (type Ia) or side-on (type Ib). Head-on anchors insert ends of stress fibers at the junctions. Apical stress fibers can link junctions from opposite side of the cell. Side-on anchors are found when apical stress fibers lie parallel to the junction. (B) Using head-on and side-on configurations, highlighted in pink, the apical stress fiber, represented by repeated and aligned synaptopodin densities, circled in blue, traces along the junction to form a ring that is structurally and mechanically part of the apical junction. Left panel shows the apical junction of the cell and the right panel shows the bottom of the cell; X-Y, Y-Z, and X-Z views are shown. Orange arrowheads mark synaptopodin densities of apical stress fibers in X-Z and Y-Z views. Red arrowheads mark synaptopodin densities of basal stress fibers in Y-Z view. The periodic spacing of synaptopodin densities are similar in apical and basal stress fibers, both have the alternating type I actomyosin organization. (C) Disintegration of periodic synaptopodin organization and formation of type II actomyosin structure, the contractomere, at the apical junction of mature junction. Left and middle panels show the very top and apical junction of the cell, and the right panel shows the bottom of the cell; X-Y, Y-Z, and X-Z views are shown. Basal stress fibers remain intact despite disassembly of apical stress fibers.
Figure Legend Snippet: Live-imaging of synaptopodin showing 2 apical actomyosin structures and basal stress fibers in MDCK cell monolayers. (A) Apical stress fibers are outlined by aligned and repeated synaptopodin densities, circled in blue, yellow, and green. Apical stress fibers are inserted at the junction via synaptopodin linkers, highlighted in pink, using 2 configurations, either head-on (type Ia) or side-on (type Ib). Head-on anchors insert ends of stress fibers at the junctions. Apical stress fibers can link junctions from opposite side of the cell. Side-on anchors are found when apical stress fibers lie parallel to the junction. (B) Using head-on and side-on configurations, highlighted in pink, the apical stress fiber, represented by repeated and aligned synaptopodin densities, circled in blue, traces along the junction to form a ring that is structurally and mechanically part of the apical junction. Left panel shows the apical junction of the cell and the right panel shows the bottom of the cell; X-Y, Y-Z, and X-Z views are shown. Orange arrowheads mark synaptopodin densities of apical stress fibers in X-Z and Y-Z views. Red arrowheads mark synaptopodin densities of basal stress fibers in Y-Z view. The periodic spacing of synaptopodin densities are similar in apical and basal stress fibers, both have the alternating type I actomyosin organization. (C) Disintegration of periodic synaptopodin organization and formation of type II actomyosin structure, the contractomere, at the apical junction of mature junction. Left and middle panels show the very top and apical junction of the cell, and the right panel shows the bottom of the cell; X-Y, Y-Z, and X-Z views are shown. Basal stress fibers remain intact despite disassembly of apical stress fibers.

Techniques Used: Imaging

Synaptopodin is localized to basal stress fibers. (A) Alternating arrangement of synaptopodin and myosin IIB forming basal stress fiber sarcomeric-like repeats. (B) Synaptopodin-associated basal stress fibers are inserted into vinculin-decorated focal adhesions.
Figure Legend Snippet: Synaptopodin is localized to basal stress fibers. (A) Alternating arrangement of synaptopodin and myosin IIB forming basal stress fiber sarcomeric-like repeats. (B) Synaptopodin-associated basal stress fibers are inserted into vinculin-decorated focal adhesions.

Techniques Used:

Three actomyosin structures failed to form in synaptopodin knockdown cells. (A) Apical stress fibers are missing in synaptopodin knockdown cells. (B) Myosin IIB is absent from the apical junction in synaptopodin knockdown cells. Graph shows intensity measurement of myosin IIB along the linear junction. Bars mark the means. (C) Correlation between synaptopodin and myosin IIB levels at the apical junction. (D) Basal stress fibers are missing in synaptopodin knockdown cells. (E) Contractomeres is present in mature MDCK monolayer. White boxes are enlarged to locate the contractomeres, white circles. (F) Contractomeres are missing in synaptopodin knockdown cells. (G) Contractomeric myosin IIB is abolished in synaptopodin knockdown cells. (H) Total cellular levels of myosin IIB and phospho-myosin light chain are lower in synaptopodin knockdown cells. Lane 1 and 3 are whole cell lysate of MDCK parental cells. Lane 2 is whole cell lysate of synaptopodin knockdown cells. Markers are 150, 100, 75, 25 kD.
Figure Legend Snippet: Three actomyosin structures failed to form in synaptopodin knockdown cells. (A) Apical stress fibers are missing in synaptopodin knockdown cells. (B) Myosin IIB is absent from the apical junction in synaptopodin knockdown cells. Graph shows intensity measurement of myosin IIB along the linear junction. Bars mark the means. (C) Correlation between synaptopodin and myosin IIB levels at the apical junction. (D) Basal stress fibers are missing in synaptopodin knockdown cells. (E) Contractomeres is present in mature MDCK monolayer. White boxes are enlarged to locate the contractomeres, white circles. (F) Contractomeres are missing in synaptopodin knockdown cells. (G) Contractomeric myosin IIB is abolished in synaptopodin knockdown cells. (H) Total cellular levels of myosin IIB and phospho-myosin light chain are lower in synaptopodin knockdown cells. Lane 1 and 3 are whole cell lysate of MDCK parental cells. Lane 2 is whole cell lysate of synaptopodin knockdown cells. Markers are 150, 100, 75, 25 kD.

Techniques Used:

Superresolution immunofluorescence microscopy of 2 actomyosin structures at the apical junction of MDCK epithelial cell monolayers. (A) Apical stress fibers are characterized by alternating pattern of synaptopodin and myosin IIB. (B) Apical stress fibers are inserted at cell-cell adhesions marked by synaptopodin and alpha-actinin-4. (C) Apical stress fibers are labelled as Type I actomyosin structure with alternating synaptopodin and myosin IIB organization. Type I apical stress fiber disintegrates upon junction maturation which coincides with the appearance of Type II actomyosin structure containing synaptopodin and myosin IIB. Basal stress fibers have alternating pattern of synaptopodin and myosin IIB organization similar to Type I apical stress fibers.
Figure Legend Snippet: Superresolution immunofluorescence microscopy of 2 actomyosin structures at the apical junction of MDCK epithelial cell monolayers. (A) Apical stress fibers are characterized by alternating pattern of synaptopodin and myosin IIB. (B) Apical stress fibers are inserted at cell-cell adhesions marked by synaptopodin and alpha-actinin-4. (C) Apical stress fibers are labelled as Type I actomyosin structure with alternating synaptopodin and myosin IIB organization. Type I apical stress fiber disintegrates upon junction maturation which coincides with the appearance of Type II actomyosin structure containing synaptopodin and myosin IIB. Basal stress fibers have alternating pattern of synaptopodin and myosin IIB organization similar to Type I apical stress fibers.

Techniques Used: Immunofluorescence, Microscopy

Tension homeostasis is compromised in synaptopodin knockdown cells. (A) Cartoon showing the balance between cellular contractility and junctional tension. (B) Cartoon illustrating how hydraulic force is used to activate junction contractility. (C) Synaptopodin knockdown increases cell extrusion and decreases alpha-actinin-4 recruitment under force. Red asterisks mark extruded cells. Graph shows increased cell extrusion in synaptopodin knockdown monolayer. Bars mark the means.
Figure Legend Snippet: Tension homeostasis is compromised in synaptopodin knockdown cells. (A) Cartoon showing the balance between cellular contractility and junctional tension. (B) Cartoon illustrating how hydraulic force is used to activate junction contractility. (C) Synaptopodin knockdown increases cell extrusion and decreases alpha-actinin-4 recruitment under force. Red asterisks mark extruded cells. Graph shows increased cell extrusion in synaptopodin knockdown monolayer. Bars mark the means.

Techniques Used:

Polarized phenotype of MDCK cells are unchanged by synaptopodin depletion. Reconstruction of Z-stacks shows that synaptopodin knockdown cells have similar overall morphology as parental MDCK cells.
Figure Legend Snippet: Polarized phenotype of MDCK cells are unchanged by synaptopodin depletion. Reconstruction of Z-stacks shows that synaptopodin knockdown cells have similar overall morphology as parental MDCK cells.

Techniques Used:

Contractomere glides on the membrane to shorten or lengthen the junction. (A) Contractomere (blue circles) glides towards each other to constrict the apical junction during apoptotic cell extrusion (orange asterisk). Frames from time-lapse wide-field movie of venus-alpha-actinin-1. Yellow arrows mark the paths of contractomere movement. Orange circle shows the location of contractomeres after constriction. (B) Contractomere (yellow circles) glides toward each other to constrict the apical junction during live-cell extrusion (white asterisk). Frames from time-lapse wide-field movie of venus-alpha-actinin-1. Live-cell extrusion is not associated with membrane blebbing of extruding cell. Yellow arrows mark the paths of contractomere movement. Orange circle shows the location of contractomeres after constriction. (C) Contractomere (circles) glides towards each other to constrict the apical junction during apoptotic cell extrusion (white asterisk). Frames from time-lapse structured-illumination movies of venus-alpha-actinin-1. Orange circle show the location of contractomeres after completion of constriction. (D) Contractomere (circles) glides towards each other to constrict the apical junction during live-cell extrusion (white asterisk). Frames from time-lapse movies of structured-illumination microscopy of venus-alpha-actinin-1. Live-cell extrusion is not associated with membrane blebbing of extruding cell. (E) Contractomeres (circles) glide to shorten the apical junction surround the extruding cell (white asterisk) while lengthen the apical junction in the neighboring cells (blue and orange arrows). Frames from time-lapse structured-illumination movies of venus-alpha-actinin-1. Live-cell extrusion is not associated with membrane blebbing of extruding cell. (F) Contractomere slides during intercellular movement. Sliding of contractomere can either shorten or lengthen the junction. Cells #1 and #2 are used to illustrate that some junctions increased but the others decreased their length, resulting in almost zero net change in total junctional length. Frames from time-lapse structured-illumination movies of venus-alpha-actinin-1. (G) Gliding of contractomeres to reproportion junctional lengths. Movement of contractomere b towards contractomere a resulted in shortening the junction between a and b with concomitant lengthening of the junction between contractomeres b and c. Frames from time-lapse wide-field movies of venus-alpha-actinin-1. (H) Gliding of contractomeres to reproportion junctional lengths. Movement of contractomere b away from contractomere a resulted in lengthening of the junction between a and b with concomitant shortening of the junction between contractomeres b and c. Frames from time-lapse wide-field movie of synaptopodin-venus.
Figure Legend Snippet: Contractomere glides on the membrane to shorten or lengthen the junction. (A) Contractomere (blue circles) glides towards each other to constrict the apical junction during apoptotic cell extrusion (orange asterisk). Frames from time-lapse wide-field movie of venus-alpha-actinin-1. Yellow arrows mark the paths of contractomere movement. Orange circle shows the location of contractomeres after constriction. (B) Contractomere (yellow circles) glides toward each other to constrict the apical junction during live-cell extrusion (white asterisk). Frames from time-lapse wide-field movie of venus-alpha-actinin-1. Live-cell extrusion is not associated with membrane blebbing of extruding cell. Yellow arrows mark the paths of contractomere movement. Orange circle shows the location of contractomeres after constriction. (C) Contractomere (circles) glides towards each other to constrict the apical junction during apoptotic cell extrusion (white asterisk). Frames from time-lapse structured-illumination movies of venus-alpha-actinin-1. Orange circle show the location of contractomeres after completion of constriction. (D) Contractomere (circles) glides towards each other to constrict the apical junction during live-cell extrusion (white asterisk). Frames from time-lapse movies of structured-illumination microscopy of venus-alpha-actinin-1. Live-cell extrusion is not associated with membrane blebbing of extruding cell. (E) Contractomeres (circles) glide to shorten the apical junction surround the extruding cell (white asterisk) while lengthen the apical junction in the neighboring cells (blue and orange arrows). Frames from time-lapse structured-illumination movies of venus-alpha-actinin-1. Live-cell extrusion is not associated with membrane blebbing of extruding cell. (F) Contractomere slides during intercellular movement. Sliding of contractomere can either shorten or lengthen the junction. Cells #1 and #2 are used to illustrate that some junctions increased but the others decreased their length, resulting in almost zero net change in total junctional length. Frames from time-lapse structured-illumination movies of venus-alpha-actinin-1. (G) Gliding of contractomeres to reproportion junctional lengths. Movement of contractomere b towards contractomere a resulted in shortening the junction between a and b with concomitant lengthening of the junction between contractomeres b and c. Frames from time-lapse wide-field movies of venus-alpha-actinin-1. (H) Gliding of contractomeres to reproportion junctional lengths. Movement of contractomere b away from contractomere a resulted in lengthening of the junction between a and b with concomitant shortening of the junction between contractomeres b and c. Frames from time-lapse wide-field movie of synaptopodin-venus.

Techniques Used: Microscopy

Contractomeric myosin II activity is linked to actin accumulation. (A) Inhibition of myosin II ATPase by blebbistatin abolished latrunculin (LatB)-resistant actin at the apical junctional complex. (B) Synaptopodin marks latrunculin-resistant pool of actin at the apical junction. (C) Inhibition of myosin II ATPase by blebbistatin (Bleb) decreased latrunculin-resistant actin accumulation in the presence of barbed-end capping by cytochalasin D (Cyto D). Graphs show intensity measurement of actin on individual contractomeres. Bars mark the means. (D) Non-invasion wound healing assay showing the formation of latrunculin-resistant actin at wound edge, circled, which is inhibited by blebbistatin (Bleb). Latrunculin-resistant actin is formed after 1-hour incubation with cytochalasin D (Cyto D) and latrunculin B (Lat B). In the absence of blebbistatin (Bleb), cytochalasin D (Cyto D) or latrunculin B (Lat B), the wound is closed in an hour. Left panels show phase-contrast images of MDCK dome before wounding.
Figure Legend Snippet: Contractomeric myosin II activity is linked to actin accumulation. (A) Inhibition of myosin II ATPase by blebbistatin abolished latrunculin (LatB)-resistant actin at the apical junctional complex. (B) Synaptopodin marks latrunculin-resistant pool of actin at the apical junction. (C) Inhibition of myosin II ATPase by blebbistatin (Bleb) decreased latrunculin-resistant actin accumulation in the presence of barbed-end capping by cytochalasin D (Cyto D). Graphs show intensity measurement of actin on individual contractomeres. Bars mark the means. (D) Non-invasion wound healing assay showing the formation of latrunculin-resistant actin at wound edge, circled, which is inhibited by blebbistatin (Bleb). Latrunculin-resistant actin is formed after 1-hour incubation with cytochalasin D (Cyto D) and latrunculin B (Lat B). In the absence of blebbistatin (Bleb), cytochalasin D (Cyto D) or latrunculin B (Lat B), the wound is closed in an hour. Left panels show phase-contrast images of MDCK dome before wounding.

Techniques Used: Activity Assay, Inhibition, Wound Healing Assay, Incubation

4) Product Images from "uPAR isoform 2 forms a dimer and induces severe kidney disease in mice"

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI124793

Glomerular c-Src activity is increased in human FSGS kidney. Immunofluorescent staining with p–c-Src antibody was performed for the frozen sections of deidentified human kidney biopsies. Synaptopodin was used as a podocyte marker. Shown is a representative of 4 batches of immunostaining. While a minimal amount of c-Src phosphorylation was observed in the glomeruli of healthy donors ( n = 3), glomerular p–c-Src intensity was increased in 6 out of 10 FSGS patients. Overlap of p–c-Src (green) and synpo (red ) indicates that p–c-Src was localized in podocytes. Of note, only 11 out of 24 observed glomeruli were positive for p–c-Src, from which 64% were focal, 36% globally but not evenly. In contrast, the increase of pSrc was not observed in other glomerular diseases, including SLE ( n = 2), MPGN ( n = 2), and MCD ( n = 4). Scale bar: 20 μm. NT, normal kidney tissue.
Figure Legend Snippet: Glomerular c-Src activity is increased in human FSGS kidney. Immunofluorescent staining with p–c-Src antibody was performed for the frozen sections of deidentified human kidney biopsies. Synaptopodin was used as a podocyte marker. Shown is a representative of 4 batches of immunostaining. While a minimal amount of c-Src phosphorylation was observed in the glomeruli of healthy donors ( n = 3), glomerular p–c-Src intensity was increased in 6 out of 10 FSGS patients. Overlap of p–c-Src (green) and synpo (red ) indicates that p–c-Src was localized in podocytes. Of note, only 11 out of 24 observed glomeruli were positive for p–c-Src, from which 64% were focal, 36% globally but not evenly. In contrast, the increase of pSrc was not observed in other glomerular diseases, including SLE ( n = 2), MPGN ( n = 2), and MCD ( n = 4). Scale bar: 20 μm. NT, normal kidney tissue.

Techniques Used: Activity Assay, Staining, Marker, Immunostaining

5) Product Images from "Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction"

Article Title: Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction

Journal: bioRxiv

doi: 10.1101/2020.12.30.424702

Contractomeric myosin II activity is linked to actin accumulation. (A) Immunofluorescence showing synaptopodin colocalizes with latrunculin-resistant actin at the apical junction. Scale bar is 2 microns. (B) Immunofluorescence showing myosin IIB colocalizes with actin puncta. Scale bar is 1 micron. (C) Myosin II inhibition prevents the formation of latrunculin (LatB)-resistant actin at the apical junction. Scale bar is 2 microns. (D) Myosin II inhibition prevents the formation of latrunculin (LatB)-resistant actin in the presence or absence of barbed-end capping by cytochalasin D (CytoD). Graphs show intensity measurement of actin on individual contractomeres. Bars mark the means. P
Figure Legend Snippet: Contractomeric myosin II activity is linked to actin accumulation. (A) Immunofluorescence showing synaptopodin colocalizes with latrunculin-resistant actin at the apical junction. Scale bar is 2 microns. (B) Immunofluorescence showing myosin IIB colocalizes with actin puncta. Scale bar is 1 micron. (C) Myosin II inhibition prevents the formation of latrunculin (LatB)-resistant actin at the apical junction. Scale bar is 2 microns. (D) Myosin II inhibition prevents the formation of latrunculin (LatB)-resistant actin in the presence or absence of barbed-end capping by cytochalasin D (CytoD). Graphs show intensity measurement of actin on individual contractomeres. Bars mark the means. P

Techniques Used: Activity Assay, Immunofluorescence, Inhibition

Contractomeres formation at late stage of junction maturation. (A) Structured-illumination microscopy of synaptopodin and ZO-1. A z-stack is shown in X-Y, Y-Z, and X-Y axes. Left panel shows contractomeres on the apical plane. Right panel shows basal stress fibers. Arrows on Y-Z axis point to the basal plane. Scale bar is 5 microns. (B) Disintegration of periodic synaptopodin organization and formation of contractomeres at the apical junction in mature monolayer. Left and middle panels show the top and apical junction , and the right panel shows the basal palne; X-Y, Y-Z, and X-Z views are shown. Basal stress fibers remain intact despite disassembly of apical stress fibers. Contractomeres are squared. Scale bar is 5 microns. (C) Live-cell Structured-illuminated microscopy of synaptopodin and vinculin. Vinculin is absent from the apical junction in developing monolayer. Blue and orange boxes are higher magnification showing basal junctions and focal adhesions on the same basal focal plane. Scale bars are 2 microns. (D) Vinculin is absent from the apical junction in mature monolayer. A z-stack is shown in X-Y, X-Z, and Y-Z views. Scale bar is 5 microns.
Figure Legend Snippet: Contractomeres formation at late stage of junction maturation. (A) Structured-illumination microscopy of synaptopodin and ZO-1. A z-stack is shown in X-Y, Y-Z, and X-Y axes. Left panel shows contractomeres on the apical plane. Right panel shows basal stress fibers. Arrows on Y-Z axis point to the basal plane. Scale bar is 5 microns. (B) Disintegration of periodic synaptopodin organization and formation of contractomeres at the apical junction in mature monolayer. Left and middle panels show the top and apical junction , and the right panel shows the basal palne; X-Y, Y-Z, and X-Z views are shown. Basal stress fibers remain intact despite disassembly of apical stress fibers. Contractomeres are squared. Scale bar is 5 microns. (C) Live-cell Structured-illuminated microscopy of synaptopodin and vinculin. Vinculin is absent from the apical junction in developing monolayer. Blue and orange boxes are higher magnification showing basal junctions and focal adhesions on the same basal focal plane. Scale bars are 2 microns. (D) Vinculin is absent from the apical junction in mature monolayer. A z-stack is shown in X-Y, X-Z, and Y-Z views. Scale bar is 5 microns.

Techniques Used: Microscopy

Contractomeres and stress fibers are missing in synaptopodin knockdown cells. (A) Immunofluorescence of MDCK wild type (WT) and synaptopodin knockdown (Synpo KD) cells. Apical and basal stress fibers are missing in synaptopodin knockdown cells. Scale bars are 1 micron. (B) Western blots showing reduced cellular levels of myosin IIB and myosin light chain in synaptopodin knockdown cells. Lanes 1 and 3 are whole cell lysate of MDCK parental cells. Lane 2 is whole cell lysate of synaptopodin knockdown cells. Markers are 150, 100, 75, 25 kD. (C) Scatter plot showing relationship between synaptopodin and myosin IIB levels in MDCK WT (red dots) and Synpo KD (blue squares) at the apical junction. Pearson correlation coefficient is 0.63 between synaptopodin and myosin IIB in WT junction. (D) Myosin IIB is absent from the apical junction in synaptopodin knockdown cells despite the presence of junctional actin. Scale bar is 5 microns. (E) Synaptopodin knockdown (Synpo KD) specifically reduced myosin IIB but not myosin IIA. Measurement of junctional and contractomeric intensities of myosin IIA and IIB immunofluorescence. P
Figure Legend Snippet: Contractomeres and stress fibers are missing in synaptopodin knockdown cells. (A) Immunofluorescence of MDCK wild type (WT) and synaptopodin knockdown (Synpo KD) cells. Apical and basal stress fibers are missing in synaptopodin knockdown cells. Scale bars are 1 micron. (B) Western blots showing reduced cellular levels of myosin IIB and myosin light chain in synaptopodin knockdown cells. Lanes 1 and 3 are whole cell lysate of MDCK parental cells. Lane 2 is whole cell lysate of synaptopodin knockdown cells. Markers are 150, 100, 75, 25 kD. (C) Scatter plot showing relationship between synaptopodin and myosin IIB levels in MDCK WT (red dots) and Synpo KD (blue squares) at the apical junction. Pearson correlation coefficient is 0.63 between synaptopodin and myosin IIB in WT junction. (D) Myosin IIB is absent from the apical junction in synaptopodin knockdown cells despite the presence of junctional actin. Scale bar is 5 microns. (E) Synaptopodin knockdown (Synpo KD) specifically reduced myosin IIB but not myosin IIA. Measurement of junctional and contractomeric intensities of myosin IIA and IIB immunofluorescence. P

Techniques Used: Immunofluorescence, Western Blot

Unconventional biochemistry for actin assembly at the contractomere. (A) Immunofluorescence of junctional membranes showing myosin IIB and synaptopodin colocalize with alpha-actinin-4 on contractomeres. Scale bar is 1 micron. (B) Myosin IIB is localized to sites of actin assembly on junction membranes. Actin is initiated by adding G-actin to purified membranes in the presence of ATP (see Methods). Immunofluorescence staining for myosin IIB was performed on membranes after an actin assembly assay. Scale bar is 500 nm. (C) Actin assembly on purified membrane is blocked by the addition of blebbistatin to the actin assembly mixture (see Methods). Scale bar is 5 microns. (D) Negative-stain electron microscopy of contractomeres after actin assembly reaction (see Methods). Upper left panels show actin bundles wrap around to form a ball (circled) after an actin assembly reaction using 2 micromolar of G-actin. Single actin filaments (arrows) can be seen in associated with the actin ball. Scale bar is 200 nm. Upper right and lower left panels showing actin filaments (yellow arrows) interact with multiple globular densities (red arrows) on the contractomeres after an actin assembly reaction using 500 nM of G-actin. Scale bars are 100 nm. Lower right panel shows a contractomeric complex extracted with CHAPS detergent to remove lipids components. Multiple densities (red arrows) are interacting with an actin filament (yellow arrows). Scale bar is 50 nm.
Figure Legend Snippet: Unconventional biochemistry for actin assembly at the contractomere. (A) Immunofluorescence of junctional membranes showing myosin IIB and synaptopodin colocalize with alpha-actinin-4 on contractomeres. Scale bar is 1 micron. (B) Myosin IIB is localized to sites of actin assembly on junction membranes. Actin is initiated by adding G-actin to purified membranes in the presence of ATP (see Methods). Immunofluorescence staining for myosin IIB was performed on membranes after an actin assembly assay. Scale bar is 500 nm. (C) Actin assembly on purified membrane is blocked by the addition of blebbistatin to the actin assembly mixture (see Methods). Scale bar is 5 microns. (D) Negative-stain electron microscopy of contractomeres after actin assembly reaction (see Methods). Upper left panels show actin bundles wrap around to form a ball (circled) after an actin assembly reaction using 2 micromolar of G-actin. Single actin filaments (arrows) can be seen in associated with the actin ball. Scale bar is 200 nm. Upper right and lower left panels showing actin filaments (yellow arrows) interact with multiple globular densities (red arrows) on the contractomeres after an actin assembly reaction using 500 nM of G-actin. Scale bars are 100 nm. Lower right panel shows a contractomeric complex extracted with CHAPS detergent to remove lipids components. Multiple densities (red arrows) are interacting with an actin filament (yellow arrows). Scale bar is 50 nm.

Techniques Used: Immunofluorescence, Purification, Staining, Electron Microscopy

Synaptopodin apical stress fibers are contractile structures distinct from basal stress fibers. (A) Frames taken from a time-lapse of synaptopodin and ZO-1 showing apical and basal stress fibers on different focal planes. Apical and basal stress fibers are inserted at apical and basal ZO-1 junctions, respectively. Scale bar is 1 micron. (B) Frames taken from a time-lapse of synaptopodin and ZO-1. Arrowheads point to sites of stress fiber attachment at the apical junction. Contraction events are circled. Dotted lines represent lengths of linear junction. Scale bar is 5 micron. (C) Frames taken from a time-lapse of synaptopodin and ZO-1. Arrowheads point to sites of stress fiber attachment at the apical junction. Contraction events are circled. Arrows point to direction of movement of the attached junction. Scale bar is 1 micron. (D) Frames taken from time-lapse of synaptopodin and ZO-1. Outline of a cell at time zero is drawn in all panels. Arrowheads point to stress fiber attachment sites at the apical junction. Contraction of apical stress fiber is circled. Scale bar is 10 microns.
Figure Legend Snippet: Synaptopodin apical stress fibers are contractile structures distinct from basal stress fibers. (A) Frames taken from a time-lapse of synaptopodin and ZO-1 showing apical and basal stress fibers on different focal planes. Apical and basal stress fibers are inserted at apical and basal ZO-1 junctions, respectively. Scale bar is 1 micron. (B) Frames taken from a time-lapse of synaptopodin and ZO-1. Arrowheads point to sites of stress fiber attachment at the apical junction. Contraction events are circled. Dotted lines represent lengths of linear junction. Scale bar is 5 micron. (C) Frames taken from a time-lapse of synaptopodin and ZO-1. Arrowheads point to sites of stress fiber attachment at the apical junction. Contraction events are circled. Arrows point to direction of movement of the attached junction. Scale bar is 1 micron. (D) Frames taken from time-lapse of synaptopodin and ZO-1. Outline of a cell at time zero is drawn in all panels. Arrowheads point to stress fiber attachment sites at the apical junction. Contraction of apical stress fiber is circled. Scale bar is 10 microns.

Techniques Used:

Live imaging of synaptopodin showing retrograde flow during early development, contraction during maturation, and anterograde flow towards junctional vertex during late stage of maturation. (A) Frames from time-lapse of synaptopodin and ZO-1 in cells with developing junctions. Retrograde synaptopodin flow originating from the apical junction. Circles track single synaptopodin densities as they flow inward from the apical junction into the medial-apical region. Scale bars are 1 micron. (B) Frames from time-lapse of synaptopodin and ZO-1 in cells with maturing junctions. ZO-1 densities, marked by arrowheads, are temporarily clustered when synaptopodin stress fiber contract (asterisk). (C) Frames from time-lapse of synaptopodin and ZO-1. Anterograde synaptopodin flow towards the junction vertex is associated with the movement of junctional vertex into the synaptopodin flow. Circles track synaptopodin densities flowing into the junction vertex. Arrowhead track the movement of junction vertex. Scale bar is 500 nm.
Figure Legend Snippet: Live imaging of synaptopodin showing retrograde flow during early development, contraction during maturation, and anterograde flow towards junctional vertex during late stage of maturation. (A) Frames from time-lapse of synaptopodin and ZO-1 in cells with developing junctions. Retrograde synaptopodin flow originating from the apical junction. Circles track single synaptopodin densities as they flow inward from the apical junction into the medial-apical region. Scale bars are 1 micron. (B) Frames from time-lapse of synaptopodin and ZO-1 in cells with maturing junctions. ZO-1 densities, marked by arrowheads, are temporarily clustered when synaptopodin stress fiber contract (asterisk). (C) Frames from time-lapse of synaptopodin and ZO-1. Anterograde synaptopodin flow towards the junction vertex is associated with the movement of junctional vertex into the synaptopodin flow. Circles track synaptopodin densities flowing into the junction vertex. Arrowhead track the movement of junction vertex. Scale bar is 500 nm.

Techniques Used: Imaging

Synaptopodin is localized to junctions and stress fibers of epithelial and endothelial cells. (A) Synaptopodin is encoded by 3 exons, resulting in 4 splice variants. We raised antibodies to each spliced region to characterize the expression of synaptopodin isoforms in mammalian tissues and cells. Western blot using antibodies against region encoded by exon 2, thus recognizing all synaptopodin isoforms. Table summarized results from western blot studies using our newly generated antibodies (see methods section). (B) Immunofluorescence of MDCK kidney tubule epithelial cells, C2bbE2 intestinal epithelial cells, HUVEC endothelial cells. Scale bars are 2 microns.
Figure Legend Snippet: Synaptopodin is localized to junctions and stress fibers of epithelial and endothelial cells. (A) Synaptopodin is encoded by 3 exons, resulting in 4 splice variants. We raised antibodies to each spliced region to characterize the expression of synaptopodin isoforms in mammalian tissues and cells. Western blot using antibodies against region encoded by exon 2, thus recognizing all synaptopodin isoforms. Table summarized results from western blot studies using our newly generated antibodies (see methods section). (B) Immunofluorescence of MDCK kidney tubule epithelial cells, C2bbE2 intestinal epithelial cells, HUVEC endothelial cells. Scale bars are 2 microns.

Techniques Used: Expressing, Western Blot, Generated, Immunofluorescence

Synaptopodin flow and the evolution of apical stress fibers during junction development. (A) Frames from time-lapse of synaptopodin and ZO-1 in developing junctions. Retrograde synaptopodin flow originates from the apical junction. Rectangles track a row of synaptopodin densities as they flow inward away from the apical junction. Circles track single synaptopodin densities. Graph shows the rate of synaptopodin flow by tracking individual synaptopodin densities. Scale bar is 2 microns. (B) Frames from time-lapse of synaptopodin and ZO-1 of maturing junctions. ZO-1 densities, marked by arrowheads, are temporarily clustered when synaptopodin stress fiber contract (circled). Scale bar is 1 micron. (C) Frames from time-lapse of synaptopodin and ZO-1 at late stage of junction maturation. Anterograde synaptopodin flow towards junction vertex is associated with the movement of vertex against the direction of synaptopodin flow. Circles track synaptopodin densities flowing into the junction vertex. Squares track the movement of junction vertex. Scale bar is 500 nm. (D) Frames from time-lapse of synaptopodin and ZO-1 at late stage of junction maturation. Synaptopodin flowing toward junction vertex is associated with movement of the vertex against the direction of synaptopodin flow. Arrowheads track synaptopodin densities flowing into junction vertex. Blue circles track the movement of junction vertex. Red and white circles track ZO-1 densities moving toward the vertex. Scale bar is 500 nm.
Figure Legend Snippet: Synaptopodin flow and the evolution of apical stress fibers during junction development. (A) Frames from time-lapse of synaptopodin and ZO-1 in developing junctions. Retrograde synaptopodin flow originates from the apical junction. Rectangles track a row of synaptopodin densities as they flow inward away from the apical junction. Circles track single synaptopodin densities. Graph shows the rate of synaptopodin flow by tracking individual synaptopodin densities. Scale bar is 2 microns. (B) Frames from time-lapse of synaptopodin and ZO-1 of maturing junctions. ZO-1 densities, marked by arrowheads, are temporarily clustered when synaptopodin stress fiber contract (circled). Scale bar is 1 micron. (C) Frames from time-lapse of synaptopodin and ZO-1 at late stage of junction maturation. Anterograde synaptopodin flow towards junction vertex is associated with the movement of vertex against the direction of synaptopodin flow. Circles track synaptopodin densities flowing into the junction vertex. Squares track the movement of junction vertex. Scale bar is 500 nm. (D) Frames from time-lapse of synaptopodin and ZO-1 at late stage of junction maturation. Synaptopodin flowing toward junction vertex is associated with movement of the vertex against the direction of synaptopodin flow. Arrowheads track synaptopodin densities flowing into junction vertex. Blue circles track the movement of junction vertex. Red and white circles track ZO-1 densities moving toward the vertex. Scale bar is 500 nm.

Techniques Used:

Apical stress fibers insert into the apical junctions of epithelial cells. (A) Immunofluorescence showing alternating arrangement of synaptopodin and myosin IIB forming sarcomeric-like repeats. Scale bar is 2 microns. (B) Immunofluorescence showing apical stress fibers inserted into cell-cell adhesions to connect multiple cells. Insets show synaptopodin and alpha-actinin-4 overlap on apical stress fibers. Scale bar is 5 microns. (C) Thin-section transmission electron microscopy showing actin bundle connected to the apical junction on one end and a sarcomeric structure on the opposite end. Scale bar is 200 nm.
Figure Legend Snippet: Apical stress fibers insert into the apical junctions of epithelial cells. (A) Immunofluorescence showing alternating arrangement of synaptopodin and myosin IIB forming sarcomeric-like repeats. Scale bar is 2 microns. (B) Immunofluorescence showing apical stress fibers inserted into cell-cell adhesions to connect multiple cells. Insets show synaptopodin and alpha-actinin-4 overlap on apical stress fibers. Scale bar is 5 microns. (C) Thin-section transmission electron microscopy showing actin bundle connected to the apical junction on one end and a sarcomeric structure on the opposite end. Scale bar is 200 nm.

Techniques Used: Immunofluorescence, Transmission Assay, Electron Microscopy

Synaptopodin knockdown reduces ArgBP2/SORBS2 levels without affecting RhoA. (A) Synaptopodin knockdown does not affect RhoA activity in MDCK cells as assessed by Rhotekin pull-down and RhoA western blot (see Methods). (B) Western blots showing decrease in ArgBP1/SORBS2 and phospho-myosin light chain levels in synaptopodin knockdown cells whereas RhoA and MRIP levels were unaffected. Markers are 100 75, 50, and 25 kD.
Figure Legend Snippet: Synaptopodin knockdown reduces ArgBP2/SORBS2 levels without affecting RhoA. (A) Synaptopodin knockdown does not affect RhoA activity in MDCK cells as assessed by Rhotekin pull-down and RhoA western blot (see Methods). (B) Western blots showing decrease in ArgBP1/SORBS2 and phospho-myosin light chain levels in synaptopodin knockdown cells whereas RhoA and MRIP levels were unaffected. Markers are 100 75, 50, and 25 kD.

Techniques Used: Activity Assay, Western Blot

Contractomere motility conserves junctional length. (A) Frames from time-lapse structured-illumination movie of venus-alpha-actinin-1 showing contractomere at the beginning (pink circles) and at the end (yellow circles) of a 4-hour movie. During intercellular organization, junctional length can shorten when 2 contractomeres move towards each other and junctional length can extend when 2 contractomeres move away from each other. The distance and direction of travel for the contractomeres are shown in upper right panel. Lower left 2 panels show the cells maintaining their neighbors during the 4-hour movie. Lower right 3 panels show contractomere motility in 2 cells. Scale bars are 10 microns. (B) Measurement of junctional lengths of the 2 cells in A. Cells #1 and #2 are used to illustrate that some junctions increased but the others decreased their length, resulting in near zero net change in total junctional length. (C) Frames from time-lapse wide-field of venus-alpha-actinin-1 and synaptopodin-venus. Gliding of contractomeres to reproportion junctional lengths. In the a-actinin movie, motility of contractomeres resulted in shortening the junction between contractomeres a and b with concomitant lengthening of the junction between contractomeres b and c. In the synaptopodin movie, motility of contractomeres resulted in lengthening of the junction between a and b with concomitant shortening of the junction between contractomeres b and c. Scale bars are 5 microns. (D) Plating of MDCK cells on collagen I at confluent density resulted in intercellular movement and neighbor exchange. Total junctional length of individual cells remained constant. Scale bar is 10 microns. (E) Frames from time-lapse of occludin showing contractomere and junction movement over 12 hours. Contractomeres are circled.
Figure Legend Snippet: Contractomere motility conserves junctional length. (A) Frames from time-lapse structured-illumination movie of venus-alpha-actinin-1 showing contractomere at the beginning (pink circles) and at the end (yellow circles) of a 4-hour movie. During intercellular organization, junctional length can shorten when 2 contractomeres move towards each other and junctional length can extend when 2 contractomeres move away from each other. The distance and direction of travel for the contractomeres are shown in upper right panel. Lower left 2 panels show the cells maintaining their neighbors during the 4-hour movie. Lower right 3 panels show contractomere motility in 2 cells. Scale bars are 10 microns. (B) Measurement of junctional lengths of the 2 cells in A. Cells #1 and #2 are used to illustrate that some junctions increased but the others decreased their length, resulting in near zero net change in total junctional length. (C) Frames from time-lapse wide-field of venus-alpha-actinin-1 and synaptopodin-venus. Gliding of contractomeres to reproportion junctional lengths. In the a-actinin movie, motility of contractomeres resulted in shortening the junction between contractomeres a and b with concomitant lengthening of the junction between contractomeres b and c. In the synaptopodin movie, motility of contractomeres resulted in lengthening of the junction between a and b with concomitant shortening of the junction between contractomeres b and c. Scale bars are 5 microns. (D) Plating of MDCK cells on collagen I at confluent density resulted in intercellular movement and neighbor exchange. Total junctional length of individual cells remained constant. Scale bar is 10 microns. (E) Frames from time-lapse of occludin showing contractomere and junction movement over 12 hours. Contractomeres are circled.

Techniques Used:

Superresolution microscopy of 2 actomyosin structures at the apical junction. (A) Apical stress fiber has alternating myosin IIB and synaptopodin pattern and lies parallel to E-cadherin junction. Orange arrowheads point to the end of an apical stress fiber marked by E-cadherin. White arrowheads point to the junctional region where synaptopodin is in proximity to E-cadherin. White long arrow shows the junctional length used for x-axis of the graph. Scale bar is 500 nm. (B) Apical stress fiber is inserted head-on at E-cadherin junction, marked by orange arrowhead. Synaptopodin overlaps with E-cadherin at the insertion point of apical stress fiber, in white circle. White arrowheads mark sites where synaptopodin is in proximity to or overlapping with E-cadherin. Scale bar is 500 nm. (C) Disassembly of apical stress fibers in maturing junction results in the loss of alternating synaptopodin and myosin IIB pattern. Disassembly of apical stress fiber coincides with the formation of type II actomyosin structures containing myosin IIB, synaptopodin, and alpha-actinin-4, marked by arrows in graph. White circles in the left panels show type II structures. White arrowheads mark the colocalization of myosin IIB, synaptopodin, alpha-actinin-4 in right panels. White long arrow shows the junctional length used for x-axis of the graph. Scale bars are 1 micron. (D) Apical stress fibers disappear upon junction maturation whereas type II actomyosin structures are prominent at mature apical junctions. Actin accumulates at type II actomyosin structure, circled in white, colocalizing with myosin IIB, synaptopodin, and alpha-actinin-4, as marked by white arrowheads. Graph shows the absence of apical stress fiber and the presence type II structure, named contractomere. White long arrow shows the junctional length used for x-axis of the graph. Scale bar is 1 micron. (E) Two actomyosin structures at the apical junction of MDCK cells. Apical stress fibers are labelled as Type I actomyosin structure with alternating synaptopodin and myosin IIB organization. In maturing junction, Type I apical stress fiber coexists with Type II contractomeres with overlapping synaptopodin and myosin IIB. Basal stress fibers have alternating synaptopodin and myosin IIB organization, same as Type I apical stress fibers. Scale bar is 2 microns.
Figure Legend Snippet: Superresolution microscopy of 2 actomyosin structures at the apical junction. (A) Apical stress fiber has alternating myosin IIB and synaptopodin pattern and lies parallel to E-cadherin junction. Orange arrowheads point to the end of an apical stress fiber marked by E-cadherin. White arrowheads point to the junctional region where synaptopodin is in proximity to E-cadherin. White long arrow shows the junctional length used for x-axis of the graph. Scale bar is 500 nm. (B) Apical stress fiber is inserted head-on at E-cadherin junction, marked by orange arrowhead. Synaptopodin overlaps with E-cadherin at the insertion point of apical stress fiber, in white circle. White arrowheads mark sites where synaptopodin is in proximity to or overlapping with E-cadherin. Scale bar is 500 nm. (C) Disassembly of apical stress fibers in maturing junction results in the loss of alternating synaptopodin and myosin IIB pattern. Disassembly of apical stress fiber coincides with the formation of type II actomyosin structures containing myosin IIB, synaptopodin, and alpha-actinin-4, marked by arrows in graph. White circles in the left panels show type II structures. White arrowheads mark the colocalization of myosin IIB, synaptopodin, alpha-actinin-4 in right panels. White long arrow shows the junctional length used for x-axis of the graph. Scale bars are 1 micron. (D) Apical stress fibers disappear upon junction maturation whereas type II actomyosin structures are prominent at mature apical junctions. Actin accumulates at type II actomyosin structure, circled in white, colocalizing with myosin IIB, synaptopodin, and alpha-actinin-4, as marked by white arrowheads. Graph shows the absence of apical stress fiber and the presence type II structure, named contractomere. White long arrow shows the junctional length used for x-axis of the graph. Scale bar is 1 micron. (E) Two actomyosin structures at the apical junction of MDCK cells. Apical stress fibers are labelled as Type I actomyosin structure with alternating synaptopodin and myosin IIB organization. In maturing junction, Type I apical stress fiber coexists with Type II contractomeres with overlapping synaptopodin and myosin IIB. Basal stress fibers have alternating synaptopodin and myosin IIB organization, same as Type I apical stress fibers. Scale bar is 2 microns.

Techniques Used: Microscopy

Vinculin marks the basal junction whereas ZO-1 marks both apical and basal junctions. (A) Structured-illumination microscopy of synaptopodin and ZO-1. A z-stack is shown in X-Y, Y-Z, and X-Y axes. Left panel shows apical stress fibers and contractomeres on the apical plane. Right panel shows basal stress fibers inserted at basal junctions. Attachment sites for synaptopodin apical stress fibers are circled. Arrows on Y-Z axis point to apical and basal planes. Scale bar is 5 microns. (B) Live-cell structured-illumination microscopy of synaptopodin and vinculin. Vinculin marks the basal junction where basal stress fibers are inserted. Inset shows synaptopodin stress fibers inserted at vinculin-decorated focal adhesions. Graph shows a line scan of the arrow along a basal stress fiber in the inset. Synaptopodin has periodic organization at the stress fiber. Scale bar is 5 microns.
Figure Legend Snippet: Vinculin marks the basal junction whereas ZO-1 marks both apical and basal junctions. (A) Structured-illumination microscopy of synaptopodin and ZO-1. A z-stack is shown in X-Y, Y-Z, and X-Y axes. Left panel shows apical stress fibers and contractomeres on the apical plane. Right panel shows basal stress fibers inserted at basal junctions. Attachment sites for synaptopodin apical stress fibers are circled. Arrows on Y-Z axis point to apical and basal planes. Scale bar is 5 microns. (B) Live-cell structured-illumination microscopy of synaptopodin and vinculin. Vinculin marks the basal junction where basal stress fibers are inserted. Inset shows synaptopodin stress fibers inserted at vinculin-decorated focal adhesions. Graph shows a line scan of the arrow along a basal stress fiber in the inset. Synaptopodin has periodic organization at the stress fiber. Scale bar is 5 microns.

Techniques Used: Microscopy

Retrograde synaptopodin flow from the basal junction. (A) Frames taken from a time-lapse of synaptopodin and ZO-1. Synaptopodin retrograde flow on opposite sides of a basal junction. Arrowheads track the flow of synaptopodin puncta in 2 cells. Graph shows rate of retrograde flow by tracking synaptopodin puncta. Scale bar is 1 micron. (B) Structured-illumination live-microscopy of synaptopodin and vinculin showing insertion of stress fibers from 2 cells at basal junctions. Scale bar is 2 microns. (C) Frames taken from a time-lapse movie of synaptopodin and vinculin. One cell is expressing synaptopodin to show retrograde synaptopodin flow from basal junctions marked by the neighboring cell. Arrowheads track the flow of synaptopodin puncta. Scale bar is 1 micron. (D) Live imaging of synaptopodin. Left panel shows the apical plane and right panel shows the basal plane of the cell; X-Y, Y-Z, and X-Z views are shown. Synaptopodin linkers at the apical junction is highlighted in purple. The repeated and aligned synaptopodin densities are circled in blue. Yellow arrowheads mark synaptopodin at apical stress fibers in X-Z and Y-Z views. Pink arrowheads mark synaptopodin at basal stress fibers in Y-Z view. The periodic spacing of synaptopodin densities are seen in both apical and basal stress fibers. Scale bar is 2 microns.
Figure Legend Snippet: Retrograde synaptopodin flow from the basal junction. (A) Frames taken from a time-lapse of synaptopodin and ZO-1. Synaptopodin retrograde flow on opposite sides of a basal junction. Arrowheads track the flow of synaptopodin puncta in 2 cells. Graph shows rate of retrograde flow by tracking synaptopodin puncta. Scale bar is 1 micron. (B) Structured-illumination live-microscopy of synaptopodin and vinculin showing insertion of stress fibers from 2 cells at basal junctions. Scale bar is 2 microns. (C) Frames taken from a time-lapse movie of synaptopodin and vinculin. One cell is expressing synaptopodin to show retrograde synaptopodin flow from basal junctions marked by the neighboring cell. Arrowheads track the flow of synaptopodin puncta. Scale bar is 1 micron. (D) Live imaging of synaptopodin. Left panel shows the apical plane and right panel shows the basal plane of the cell; X-Y, Y-Z, and X-Z views are shown. Synaptopodin linkers at the apical junction is highlighted in purple. The repeated and aligned synaptopodin densities are circled in blue. Yellow arrowheads mark synaptopodin at apical stress fibers in X-Z and Y-Z views. Pink arrowheads mark synaptopodin at basal stress fibers in Y-Z view. The periodic spacing of synaptopodin densities are seen in both apical and basal stress fibers. Scale bar is 2 microns.

Techniques Used: Microscopy, Expressing, Imaging

6) Product Images from "Angiopoietins Modulate Endothelial Adaptation, Glomerular and Podocyte Hypertrophy after Uninephrectomy"

Article Title: Angiopoietins Modulate Endothelial Adaptation, Glomerular and Podocyte Hypertrophy after Uninephrectomy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082592

Podocyte evaluation at 3 months after uninephrectomy. (A) Immunofluorescence study of glomerular synaptopodin and WT1 in each group. (B) Average glomerular podocyte number (evaluated using the number of WT1-positive staining nuclei) in each group. No differences were noted among the groups. (C) Western blotting for glomerular WT1 protein expression in each group. No differences in WT1 expression were observed among the groups. (D) Podocyte hypertrophy index in each group. Uninephrectomy induced significant podocyte hypertrophy, while the administration of the Angpt 1 antagonist attenuated the degree of hypertrophy, while blocking Angpt 2 action only tended to attenuate hypertrophy. (E) Minor foot process width measured by electron microscopy. Uninephrectomy did not influence podocyte foot process width. The administration of Angpt 1 or Angpt 2 antagonists significantly decreased the podocyte foot process width in uninephrectomized mice (* P
Figure Legend Snippet: Podocyte evaluation at 3 months after uninephrectomy. (A) Immunofluorescence study of glomerular synaptopodin and WT1 in each group. (B) Average glomerular podocyte number (evaluated using the number of WT1-positive staining nuclei) in each group. No differences were noted among the groups. (C) Western blotting for glomerular WT1 protein expression in each group. No differences in WT1 expression were observed among the groups. (D) Podocyte hypertrophy index in each group. Uninephrectomy induced significant podocyte hypertrophy, while the administration of the Angpt 1 antagonist attenuated the degree of hypertrophy, while blocking Angpt 2 action only tended to attenuate hypertrophy. (E) Minor foot process width measured by electron microscopy. Uninephrectomy did not influence podocyte foot process width. The administration of Angpt 1 or Angpt 2 antagonists significantly decreased the podocyte foot process width in uninephrectomized mice (* P

Techniques Used: Immunofluorescence, Staining, Western Blot, Expressing, Blocking Assay, Electron Microscopy, Mouse Assay

7) Product Images from "Increased long noncoding RNA maternally expressed gene 3 contributes to podocyte injury induced by high glucose through regulation of mitochondrial fission"

Article Title: Increased long noncoding RNA maternally expressed gene 3 contributes to podocyte injury induced by high glucose through regulation of mitochondrial fission

Journal: Cell Death & Disease

doi: 10.1038/s41419-020-03022-7

Knockout of lncRNA Meg3 attenuates high glucose-induced mitochondrial fission and cell injury in human podocytes. a Schematic diagram of target strategy of Meg3 knockout in human podocytes using the double-nickase/CRISPR-Cas9 System. Two pairs guide RNAs targeted to the promoter region and exon 3 of lncRNA Meg3, and the target sequence is approximately 3500 bp. b Expression of lncRNA Meg3 in human podocytes of different groups. c Expression of Nephrin and Synaptopodin in human podocytes of different groups. d Representative structured of mitochondria of human podocytes were stained by MitoTracker Deep Red and imaged by laser scanning confocal microscopy (original magnification, ×630; scale bar, 10 μm). e Quantitative analyses of expression of Nephrin and Synaptopodin in human podocytes of different groups. f Quantitative analyses of mitochondrial organization in human podocytes of different groups (mean branch length, mean number of branches per network, and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. * P
Figure Legend Snippet: Knockout of lncRNA Meg3 attenuates high glucose-induced mitochondrial fission and cell injury in human podocytes. a Schematic diagram of target strategy of Meg3 knockout in human podocytes using the double-nickase/CRISPR-Cas9 System. Two pairs guide RNAs targeted to the promoter region and exon 3 of lncRNA Meg3, and the target sequence is approximately 3500 bp. b Expression of lncRNA Meg3 in human podocytes of different groups. c Expression of Nephrin and Synaptopodin in human podocytes of different groups. d Representative structured of mitochondria of human podocytes were stained by MitoTracker Deep Red and imaged by laser scanning confocal microscopy (original magnification, ×630; scale bar, 10 μm). e Quantitative analyses of expression of Nephrin and Synaptopodin in human podocytes of different groups. f Quantitative analyses of mitochondrial organization in human podocytes of different groups (mean branch length, mean number of branches per network, and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. * P

Techniques Used: Knock-Out, CRISPR, Sequencing, Expressing, Staining, Confocal Microscopy

Specific knockdown of lncRNA Meg3 in podocytes alleviated Drp1 and its phosphorylation of Ser600 in podocyte from in STZ-induced diabetic mice. a LncRNA Meg3 expression in primary podocytes isolated from mice kidneys. b Synaptopodin mRNA expression in primary podocytes isolated from mice kidneys. c Nephrin mRNA expression in primary podocytes isolated from mice kidneys. d Drp1 mRNA expression in primary podocytes isolated from mice kidneys. e Immunofluorescence staining for pDrp1 (Ser600) (green) and Synaptopodin (red) in the paraffin-embedded kidney sections was performed (original magnification, ×630, scale bar, 20 μm). Data were shown as mean ± SD in all statistical graphs ( n = 6 mice per group). ** P
Figure Legend Snippet: Specific knockdown of lncRNA Meg3 in podocytes alleviated Drp1 and its phosphorylation of Ser600 in podocyte from in STZ-induced diabetic mice. a LncRNA Meg3 expression in primary podocytes isolated from mice kidneys. b Synaptopodin mRNA expression in primary podocytes isolated from mice kidneys. c Nephrin mRNA expression in primary podocytes isolated from mice kidneys. d Drp1 mRNA expression in primary podocytes isolated from mice kidneys. e Immunofluorescence staining for pDrp1 (Ser600) (green) and Synaptopodin (red) in the paraffin-embedded kidney sections was performed (original magnification, ×630, scale bar, 20 μm). Data were shown as mean ± SD in all statistical graphs ( n = 6 mice per group). ** P

Techniques Used: Mouse Assay, Expressing, Isolation, Immunofluorescence, Staining

Overexpression of lncRNA Meg3 aggravated high glucose-induced mitochondrial fission and cell injuries in human podocytes. a Expression of lncRNA Meg3 in Meg3 overexpression podocytes compared with the control. b Expression of Nephrin and Synaptopodin in human podocytes of different groups. c Representative structured of mitochondria of human podocytes were stained by MitoTracker Deep Red and imaged by laser scanning confocal microscopy (original magnification, ×630; scale bar, 10 μm). d Quantitative analyses of expression of Nephrin and Synaptopodin in human podocytes of different groups. e Quantitative analyses of mitochondrial organization on human podocytes from different groups (mean branch length, mean number of branches per network and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. * P
Figure Legend Snippet: Overexpression of lncRNA Meg3 aggravated high glucose-induced mitochondrial fission and cell injuries in human podocytes. a Expression of lncRNA Meg3 in Meg3 overexpression podocytes compared with the control. b Expression of Nephrin and Synaptopodin in human podocytes of different groups. c Representative structured of mitochondria of human podocytes were stained by MitoTracker Deep Red and imaged by laser scanning confocal microscopy (original magnification, ×630; scale bar, 10 μm). d Quantitative analyses of expression of Nephrin and Synaptopodin in human podocytes of different groups. e Quantitative analyses of mitochondrial organization on human podocytes from different groups (mean branch length, mean number of branches per network and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. * P

Techniques Used: Over Expression, Expressing, Staining, Confocal Microscopy

Generation of podocyte-specific knockdown of LncRNA Meg3 mice. a Schematic diagram of target strategy of Meg3 deficiency in podocytes using the Cre-loxP system. b The breeding strategy to generate podocyte-specific Meg3-deficient mice ( Cre + Meg3 fl/fl mice) and the actual appearance of Meg3 +/+ mice and Cre + Meg3 fl/fl mice were shown. c PCR genotyping of Meg3 +/+ mice and Cre + Meg3 fl/fl mice by tail RNA at the age of 3 weeks. d Representative images of immunofluorescence (IF) staining for Synaptopodin (green) and fluorescence in situ hybridization (FISH) for Meg3 (red) in glomeruli from Meg3 +/+ mice and Cre + Meg3 fl/fl mice (original magnification, ×630, scale bar, 20 μm) ( n = 6 mice per group). e Total RNA was extracted from primary podocytes isolated from Cre + Meg3 fl/fl mice and Meg3 +/+ mice, and real-time PCR was performed for Meg3 expression ( n = 6 mice per group). Data were presented as mean ± SD in all statistical graphs ( n = 6 mice per group). **** P
Figure Legend Snippet: Generation of podocyte-specific knockdown of LncRNA Meg3 mice. a Schematic diagram of target strategy of Meg3 deficiency in podocytes using the Cre-loxP system. b The breeding strategy to generate podocyte-specific Meg3-deficient mice ( Cre + Meg3 fl/fl mice) and the actual appearance of Meg3 +/+ mice and Cre + Meg3 fl/fl mice were shown. c PCR genotyping of Meg3 +/+ mice and Cre + Meg3 fl/fl mice by tail RNA at the age of 3 weeks. d Representative images of immunofluorescence (IF) staining for Synaptopodin (green) and fluorescence in situ hybridization (FISH) for Meg3 (red) in glomeruli from Meg3 +/+ mice and Cre + Meg3 fl/fl mice (original magnification, ×630, scale bar, 20 μm) ( n = 6 mice per group). e Total RNA was extracted from primary podocytes isolated from Cre + Meg3 fl/fl mice and Meg3 +/+ mice, and real-time PCR was performed for Meg3 expression ( n = 6 mice per group). Data were presented as mean ± SD in all statistical graphs ( n = 6 mice per group). **** P

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Isolation, Real-time Polymerase Chain Reaction, Expressing

8) Product Images from "Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms"

Article Title: Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms

Journal: Molecules

doi: 10.3390/molecules27072109

( A ) Expression of podocyte-specific markers. Undifferentiated podocytes (upper panels) were cultured at 33 °C (permissive temperature). Differentiated podocytes (lower panels) were cultured at 37 °C (non-permissive temperature) for 14 days. Representative immunofluorescence images for podocin (green dots; arrows) and synaptopodin (green line; arrows) are shown. Podocin and synaptopodin were stained with an anti-podocin antibody and an anti-synaptopodin antibody, respectively. The nucleus was stained with Hoechst 33342 (blue color). F-actin filaments were stained with Alexa Fluor TM 568 phalloidin (with the appearance of red lines). ( B ) Concentration-dependent response of palmitic acid (PA)–induced podocyte death. Podocytes were cultured in RPMI 1640 medium containing PA at concentrations between 300 and 800 µM for 24 h. Podocytes were incubated with 600 µM PA or hydrogen peroxide (H 2 O 2 ) for 24 h. N -acetylcysteine (NAC) (10 mM), a glutathione precursor, was added with PA or H 2 O 2 . ( C ) Effect of PA on podocyte viability and mechanism of PA-induced podocyte death. Cell viability was measured with the MTT assay. The data are expressed as the mean ± standard error of the mean ( n = 3–6). NS, non-significant; ** p
Figure Legend Snippet: ( A ) Expression of podocyte-specific markers. Undifferentiated podocytes (upper panels) were cultured at 33 °C (permissive temperature). Differentiated podocytes (lower panels) were cultured at 37 °C (non-permissive temperature) for 14 days. Representative immunofluorescence images for podocin (green dots; arrows) and synaptopodin (green line; arrows) are shown. Podocin and synaptopodin were stained with an anti-podocin antibody and an anti-synaptopodin antibody, respectively. The nucleus was stained with Hoechst 33342 (blue color). F-actin filaments were stained with Alexa Fluor TM 568 phalloidin (with the appearance of red lines). ( B ) Concentration-dependent response of palmitic acid (PA)–induced podocyte death. Podocytes were cultured in RPMI 1640 medium containing PA at concentrations between 300 and 800 µM for 24 h. Podocytes were incubated with 600 µM PA or hydrogen peroxide (H 2 O 2 ) for 24 h. N -acetylcysteine (NAC) (10 mM), a glutathione precursor, was added with PA or H 2 O 2 . ( C ) Effect of PA on podocyte viability and mechanism of PA-induced podocyte death. Cell viability was measured with the MTT assay. The data are expressed as the mean ± standard error of the mean ( n = 3–6). NS, non-significant; ** p

Techniques Used: Expressing, Cell Culture, Immunofluorescence, Staining, Concentration Assay, Incubation, MTT Assay

9) Product Images from "Deiodinase-3 is a thyrostat to regulate podocyte homeostasis"

Article Title: Deiodinase-3 is a thyrostat to regulate podocyte homeostasis

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2021.103617

LPS induced proteinuria is more severe in podocyte-specific D3 knockout mice (D3KO). (a) Confocal micrographs of 10-week-old mouse kidney showing decreased expression of D3 in glomeruli 24 h post LPS injection (10 mg/kg), identified by Synaptopodin stain. Left column, grayscale of α-D3; middle column, grayscale of α-Synaptopodin; right column, merge of α-D3 in green, α-Synaptopodin in red, DAPI in blue. Bar=10 µm. (b) Quantification of glomerular D3 intensity, Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 . (c) qPCR analysis of DIO3 mRNA expression in isolated glomeruli from mice treated with LPS at for indicated time points, Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 . (d) Graph depicting Albumin (mg): Creatinine (g) ratio (ACR) measured from urine of 10-week-old D3KO mice ( n=31 ) or littermate controls ( n=11 ) 24 h post intraperitoneal (i.p) injection of LPS (5 mg/kg), Non-parametric two-tailed Students t -test was used to calculate the significance, * p≤0.05 . (e) Representative TEM micrographs of glomerular capillary loops of mice 24 h post LPS injection imaged at 5000 × and 15,000 × magnifications. FP effacement is shown in arrows. (f) FP effacement was quantified by counting the number of FPs per µm GBM in each of the samples, Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 .
Figure Legend Snippet: LPS induced proteinuria is more severe in podocyte-specific D3 knockout mice (D3KO). (a) Confocal micrographs of 10-week-old mouse kidney showing decreased expression of D3 in glomeruli 24 h post LPS injection (10 mg/kg), identified by Synaptopodin stain. Left column, grayscale of α-D3; middle column, grayscale of α-Synaptopodin; right column, merge of α-D3 in green, α-Synaptopodin in red, DAPI in blue. Bar=10 µm. (b) Quantification of glomerular D3 intensity, Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 . (c) qPCR analysis of DIO3 mRNA expression in isolated glomeruli from mice treated with LPS at for indicated time points, Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 . (d) Graph depicting Albumin (mg): Creatinine (g) ratio (ACR) measured from urine of 10-week-old D3KO mice ( n=31 ) or littermate controls ( n=11 ) 24 h post intraperitoneal (i.p) injection of LPS (5 mg/kg), Non-parametric two-tailed Students t -test was used to calculate the significance, * p≤0.05 . (e) Representative TEM micrographs of glomerular capillary loops of mice 24 h post LPS injection imaged at 5000 × and 15,000 × magnifications. FP effacement is shown in arrows. (f) FP effacement was quantified by counting the number of FPs per µm GBM in each of the samples, Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 .

Techniques Used: Knock-Out, Mouse Assay, Expressing, Injection, Staining, Two Tailed Test, Real-time Polymerase Chain Reaction, Isolation, Transmission Electron Microscopy

10) Product Images from "Quercetin Attenuates Podocyte Apoptosis of Diabetic Nephropathy Through Targeting EGFR Signaling"

Article Title: Quercetin Attenuates Podocyte Apoptosis of Diabetic Nephropathy Through Targeting EGFR Signaling

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.792777

Effects of quercetin on HG-induced podocyte injury through the EGFR pathway. (A , B , G , H) Distribution and expression of synaptopodin and nephrin in podocyte through immunofluorescence. (C , D , I , J) Statistical analysis of synaptopodin, nephrin expression. (E , K) Expression of synaptopodin through western blotting. (F , L) Statistical analysis of synaptopodin protein expression. Cells were starved for 24 h and treated with normal glucose, high glucose, DMSO, quercetin, AG1478 or Q40 + AG1478 for 24 h. Data were expressed as mean ± SEM, n = 3. ## p
Figure Legend Snippet: Effects of quercetin on HG-induced podocyte injury through the EGFR pathway. (A , B , G , H) Distribution and expression of synaptopodin and nephrin in podocyte through immunofluorescence. (C , D , I , J) Statistical analysis of synaptopodin, nephrin expression. (E , K) Expression of synaptopodin through western blotting. (F , L) Statistical analysis of synaptopodin protein expression. Cells were starved for 24 h and treated with normal glucose, high glucose, DMSO, quercetin, AG1478 or Q40 + AG1478 for 24 h. Data were expressed as mean ± SEM, n = 3. ## p

Techniques Used: Expressing, Immunofluorescence, Western Blot

11) Product Images from "Astragaloside IV ameliorates diabetic nephropathy in db/db mice by inhibiting NLRP3 inflammasome-mediated inflammation"

Article Title: Astragaloside IV ameliorates diabetic nephropathy in db/db mice by inhibiting NLRP3 inflammasome-mediated inflammation

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2021.4996

AS-IV mitigates NLRP3 inflammasome activation in podocytes of the glomeruli of db/db mice. Colocalization of (A) NLRP3 with synaptopodin and (B) caspase-1 with synaptopodin (a podocyte marker) in the glomeruli of WT control mice, and vehicle- and AS-IV-treated mice (scale bar, 20 µ m; magnification, ×200). Mean fluorescence intensity of (C) NLRP3 with synaptopodin and (D) caspase-1 with synaptopodin in the glomeruli. Data are presented as the mean ± standard deviation (n=3). *** P
Figure Legend Snippet: AS-IV mitigates NLRP3 inflammasome activation in podocytes of the glomeruli of db/db mice. Colocalization of (A) NLRP3 with synaptopodin and (B) caspase-1 with synaptopodin (a podocyte marker) in the glomeruli of WT control mice, and vehicle- and AS-IV-treated mice (scale bar, 20 µ m; magnification, ×200). Mean fluorescence intensity of (C) NLRP3 with synaptopodin and (D) caspase-1 with synaptopodin in the glomeruli. Data are presented as the mean ± standard deviation (n=3). *** P

Techniques Used: Activation Assay, Mouse Assay, Marker, Fluorescence, Standard Deviation

Benazepril and AS-IV ameliorate kidney lesions and podocyte injury in db/db mice. Representative images of the pathological structure of the kidneys after (A) H E, PAS, and Masson's trichrome staining (scale bar, 20 µ m; magnification, ×200). Quantitative analyses of the results for (B) mean glomerular volume and (C) fibrotic area in the glomeruli. (D) Representative images of the ultrastructure observed using TEM (scale, 500 nm; magnification, ×15,000). (E) Quantification of GBM thickness and (F) number of podocyte foot processes per µ m GBM. (G) Representative immunofluorometric images of synaptopodin and podocin (scale, 20 µ m; magnification, ×200). Quantification of the (H) relative expression of synaptopodin and (I) podocin in the glomeruli. Data are presented as the mean ± standard deviation (n=3). * P
Figure Legend Snippet: Benazepril and AS-IV ameliorate kidney lesions and podocyte injury in db/db mice. Representative images of the pathological structure of the kidneys after (A) H E, PAS, and Masson's trichrome staining (scale bar, 20 µ m; magnification, ×200). Quantitative analyses of the results for (B) mean glomerular volume and (C) fibrotic area in the glomeruli. (D) Representative images of the ultrastructure observed using TEM (scale, 500 nm; magnification, ×15,000). (E) Quantification of GBM thickness and (F) number of podocyte foot processes per µ m GBM. (G) Representative immunofluorometric images of synaptopodin and podocin (scale, 20 µ m; magnification, ×200). Quantification of the (H) relative expression of synaptopodin and (I) podocin in the glomeruli. Data are presented as the mean ± standard deviation (n=3). * P

Techniques Used: Mouse Assay, Staining, Transmission Electron Microscopy, Expressing, Standard Deviation

12) Product Images from "Phosphorodiamidate morpholino targeting the 5′ untranslated region of the ZIKV RNA inhibits virus replication"

Article Title: Phosphorodiamidate morpholino targeting the 5′ untranslated region of the ZIKV RNA inhibits virus replication

Journal: Virology

doi: 10.1016/j.virol.2018.04.001

A. Immunostaining of ZIKV infected podocytes after treatment with DWK-1. Immunofluorescent staining of ZIKV infected podocytes using the 4G-2 antibody specific to the E protein of ZIKV. ( 1 ) Mock infected podocytes stained with 4G-2 antibody, ( 2 ) Podocytes infected with wildtype ZIKV for 72 h and stained with the 4G-2 antibody, ( 3 ) Podocytes pretreated with DWK-1 for 24 h, rinsed and infected with ZIKV for 72 h were stained with the 4G-2 antibody. ( 4 ) Isotype control for the 4G-2 antibody. Fluorescent images were taken on a Nikon TE2000S microscope mounted with a CCD camera at 200 × magnification. Nuclei (blue) were stained with DAPI. B. DWK-1 inhibits expression of E protein in ZIKV-infected podocytes . Western blot analysis of protein lysates from uninfected and ZIKV infected podocytes. Control protein lysates were prepared from mock infected podocytes and podocytes pretreated for 24 h with 10 μM DWK-1 or Co DWK-1, rinsed and cultured for additional 72 h without added morpholinos. Untreated podocytes or cells pretreated for 24 h with DWK-1 or Co DWK-1 were subsequently infected with ZIKV and protein lysates were prepared 72 h after ZIKV infection. The ZIKV expression of the E protein (E2 antigen) is shown in the top panel. The middle panel shows the podocyte biomarker Synaptopodin and the bottom panel shows GAPDH as a loading control.
Figure Legend Snippet: A. Immunostaining of ZIKV infected podocytes after treatment with DWK-1. Immunofluorescent staining of ZIKV infected podocytes using the 4G-2 antibody specific to the E protein of ZIKV. ( 1 ) Mock infected podocytes stained with 4G-2 antibody, ( 2 ) Podocytes infected with wildtype ZIKV for 72 h and stained with the 4G-2 antibody, ( 3 ) Podocytes pretreated with DWK-1 for 24 h, rinsed and infected with ZIKV for 72 h were stained with the 4G-2 antibody. ( 4 ) Isotype control for the 4G-2 antibody. Fluorescent images were taken on a Nikon TE2000S microscope mounted with a CCD camera at 200 × magnification. Nuclei (blue) were stained with DAPI. B. DWK-1 inhibits expression of E protein in ZIKV-infected podocytes . Western blot analysis of protein lysates from uninfected and ZIKV infected podocytes. Control protein lysates were prepared from mock infected podocytes and podocytes pretreated for 24 h with 10 μM DWK-1 or Co DWK-1, rinsed and cultured for additional 72 h without added morpholinos. Untreated podocytes or cells pretreated for 24 h with DWK-1 or Co DWK-1 were subsequently infected with ZIKV and protein lysates were prepared 72 h after ZIKV infection. The ZIKV expression of the E protein (E2 antigen) is shown in the top panel. The middle panel shows the podocyte biomarker Synaptopodin and the bottom panel shows GAPDH as a loading control.

Techniques Used: Immunostaining, Infection, Staining, Microscopy, Expressing, Western Blot, Cell Culture, Biomarker Assay

13) Product Images from "Quercetin Attenuates Podocyte Apoptosis of Diabetic Nephropathy Through Targeting EGFR Signaling"

Article Title: Quercetin Attenuates Podocyte Apoptosis of Diabetic Nephropathy Through Targeting EGFR Signaling

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.792777

Effects of quercetin on HG-induced podocyte injury through the EGFR pathway. (A , B , G , H) Distribution and expression of synaptopodin and nephrin in podocyte through immunofluorescence. (C , D , I , J) Statistical analysis of synaptopodin, nephrin expression. (E , K) Expression of synaptopodin through western blotting. (F , L) Statistical analysis of synaptopodin protein expression. Cells were starved for 24 h and treated with normal glucose, high glucose, DMSO, quercetin, AG1478 or Q40 + AG1478 for 24 h. Data were expressed as mean ± SEM, n = 3. ## p
Figure Legend Snippet: Effects of quercetin on HG-induced podocyte injury through the EGFR pathway. (A , B , G , H) Distribution and expression of synaptopodin and nephrin in podocyte through immunofluorescence. (C , D , I , J) Statistical analysis of synaptopodin, nephrin expression. (E , K) Expression of synaptopodin through western blotting. (F , L) Statistical analysis of synaptopodin protein expression. Cells were starved for 24 h and treated with normal glucose, high glucose, DMSO, quercetin, AG1478 or Q40 + AG1478 for 24 h. Data were expressed as mean ± SEM, n = 3. ## p

Techniques Used: Expressing, Immunofluorescence, Western Blot

14) Product Images from "BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes"

Article Title: BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

Journal: bioRxiv

doi: 10.1101/518597

MAPT phosphorylated at Ser262 increased in neuronal processes when BAG3 or SYNPO was knocked down in mature neurons. ( A ) Representative blots of MAPT and phosphorylated MAPT (p-Thr231, p-Ser262 and p-Ser396/Ser404) in neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. ( B ) Quantitation of the levels of MAPT or phosphorylated MAPT in BAG3 or SYNPO knockdown neurons from 3 independent experiments. Data were normalized to the loading control ACTB and then compared to scramble controls. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *, p
Figure Legend Snippet: MAPT phosphorylated at Ser262 increased in neuronal processes when BAG3 or SYNPO was knocked down in mature neurons. ( A ) Representative blots of MAPT and phosphorylated MAPT (p-Thr231, p-Ser262 and p-Ser396/Ser404) in neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. ( B ) Quantitation of the levels of MAPT or phosphorylated MAPT in BAG3 or SYNPO knockdown neurons from 3 independent experiments. Data were normalized to the loading control ACTB and then compared to scramble controls. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *, p

Techniques Used: Transduction, Quantitation Assay

Phosphorylated MAPT Ser262 accumulates in autophagosomes at post-synaptic densities when either BAG3 or SYNPO expression is decreased. ( A ) Representative images of LC3B and DLG4/PSD95 colocalization in dendrites. Neurons were treated with either DMSO or bafilomycin A1 (BafA1) for 4 h before fixing and immunostaining. ( B ) Representative images of p-Ser262 and DLG4 co-staining in dendrites of neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. Arrow heads denote the overlapping of fluorescence. ( C ) Quantification of colocalization using Mander’s colocalization coefficient and object based analysis. In each condition, 18-20 neurons from 2 independent experiments were counted. 1-3 processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p
Figure Legend Snippet: Phosphorylated MAPT Ser262 accumulates in autophagosomes at post-synaptic densities when either BAG3 or SYNPO expression is decreased. ( A ) Representative images of LC3B and DLG4/PSD95 colocalization in dendrites. Neurons were treated with either DMSO or bafilomycin A1 (BafA1) for 4 h before fixing and immunostaining. ( B ) Representative images of p-Ser262 and DLG4 co-staining in dendrites of neurons transduced with scramble (scr), shBag3 or shSynpo lentivirus. Arrow heads denote the overlapping of fluorescence. ( C ) Quantification of colocalization using Mander’s colocalization coefficient and object based analysis. In each condition, 18-20 neurons from 2 independent experiments were counted. 1-3 processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p

Techniques Used: Expressing, Immunostaining, Staining, Transduction, Fluorescence

BAG3 interacts with SYNPO in mature neurons. ( A ) Immunoprecipitation of endogenous BAG3 from mature rat cortical neuronal lysates. SYNPO was detected in the isolated bound fractions. ( B ) Immunoprecipitation of endogenous SYNPO from mature rat cortical neurons. Both BAG3 and SQSTM1 were detected in the precipitated fraction. ( C ) Co-immunoprecipitation of SQSTM1 and SYNPO is independent of BAG3 in mature rat neurons.
Figure Legend Snippet: BAG3 interacts with SYNPO in mature neurons. ( A ) Immunoprecipitation of endogenous BAG3 from mature rat cortical neuronal lysates. SYNPO was detected in the isolated bound fractions. ( B ) Immunoprecipitation of endogenous SYNPO from mature rat cortical neurons. Both BAG3 and SQSTM1 were detected in the precipitated fraction. ( C ) Co-immunoprecipitation of SQSTM1 and SYNPO is independent of BAG3 in mature rat neurons.

Techniques Used: Immunoprecipitation, Isolation

Loss of BAG3 or SYNPO does not affect the initiation or maturation of autophagosomes. ( A ) Schematic representation of proteinase K (PK) protection assay. This panel was adapted and reproduced from [ 46 ]. ( B ) Autophagic cargo receptor SQSTM1 was protected from PK digestion unless the detergent Triton X-100 (TX-100) was present. Lysates from scramble (scr), BAG3 or SYNPO knockdown neurons treated with or without 10 μM chloroquine (CQ) were subjected to PK protection assays. VPS18 was used as a cytosolic control. ( C ) Quantification of the amount of PK protected SQSTM1 in each condition. Percentage of PK protected SQSTM1 was the ratio of SQSTM1 in the presence PK but in the absence of TX-100 relative to its untreated control in a given condition. Data are shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test. **, p
Figure Legend Snippet: Loss of BAG3 or SYNPO does not affect the initiation or maturation of autophagosomes. ( A ) Schematic representation of proteinase K (PK) protection assay. This panel was adapted and reproduced from [ 46 ]. ( B ) Autophagic cargo receptor SQSTM1 was protected from PK digestion unless the detergent Triton X-100 (TX-100) was present. Lysates from scramble (scr), BAG3 or SYNPO knockdown neurons treated with or without 10 μM chloroquine (CQ) were subjected to PK protection assays. VPS18 was used as a cytosolic control. ( C ) Quantification of the amount of PK protected SQSTM1 in each condition. Percentage of PK protected SQSTM1 was the ratio of SQSTM1 in the presence PK but in the absence of TX-100 relative to its untreated control in a given condition. Data are shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test. **, p

Techniques Used:

Colocalization of SYNPO with BAG3, SQSTM1 and HSPA/HSP70. Cortical neurons were immunostained for SYNPO and BAG3, SQSTM1, or HSPA/HSP70. Immunofluorescence of SYNPO overlaps with BAG3, SQSTM1 or HSPA/HSP70 in neuronal processes ( A ) and soma ( B ). Corresponding line scans are shown on the right; arrowheads indicate the areas of overlapping of intensity. Quantification of colocalization using Pearson’s correlation coefficient ( C ) and object-based analysis ( D ). In each condition, 10-30 neurons from 3 independent experiments were used for quantification. Data were plotted as mean ± SEM. As MAP2 appears in a continuous localization within neuronal dendrites and barely overlaps with SYNPO (see also Figure S1 ), the colocalization between SYNPO and BAG3, SQSTM1 and HSPA/HSP70, respectively, was compared to SYNPO and MAP2 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p
Figure Legend Snippet: Colocalization of SYNPO with BAG3, SQSTM1 and HSPA/HSP70. Cortical neurons were immunostained for SYNPO and BAG3, SQSTM1, or HSPA/HSP70. Immunofluorescence of SYNPO overlaps with BAG3, SQSTM1 or HSPA/HSP70 in neuronal processes ( A ) and soma ( B ). Corresponding line scans are shown on the right; arrowheads indicate the areas of overlapping of intensity. Quantification of colocalization using Pearson’s correlation coefficient ( C ) and object-based analysis ( D ). In each condition, 10-30 neurons from 3 independent experiments were used for quantification. Data were plotted as mean ± SEM. As MAP2 appears in a continuous localization within neuronal dendrites and barely overlaps with SYNPO (see also Figure S1 ), the colocalization between SYNPO and BAG3, SQSTM1 and HSPA/HSP70, respectively, was compared to SYNPO and MAP2 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p

Techniques Used: Immunofluorescence

Loss of BAG3 or SYNPO reduces LC3B-II and SQSTM1 turnover. ( A ) LC3B-I/II levels in primary cortical neurons transduced with lentivirus expressing shBag3 or a scrambled (scr) version. Neurons were treated with or without 10 μM chloroquine (CQ) for 16h. ( B ) LC3B blots of neurons transduced with lentivirus expressing shRNA for rat Synpo or a scrambled ( scr ) version. Neurons were treated as ( A ). ( C ) Quantifications of LC3B-II in BAG3 and SYNPO knockdown neurons in the absence or presence of CQ treatment. LC3B-II was normalized to the loading control ACTB then compared to the scrambled condition. Graph show mean ± SEM of 4-6 samples from 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *, p
Figure Legend Snippet: Loss of BAG3 or SYNPO reduces LC3B-II and SQSTM1 turnover. ( A ) LC3B-I/II levels in primary cortical neurons transduced with lentivirus expressing shBag3 or a scrambled (scr) version. Neurons were treated with or without 10 μM chloroquine (CQ) for 16h. ( B ) LC3B blots of neurons transduced with lentivirus expressing shRNA for rat Synpo or a scrambled ( scr ) version. Neurons were treated as ( A ). ( C ) Quantifications of LC3B-II in BAG3 and SYNPO knockdown neurons in the absence or presence of CQ treatment. LC3B-II was normalized to the loading control ACTB then compared to the scrambled condition. Graph show mean ± SEM of 4-6 samples from 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *, p

Techniques Used: Transduction, Expressing, shRNA

Colocalization of BAG3, SYNPO or SQSTM1 with endogenous MAP1LC3B/LC3B in neuronal processes. ( A ) Neurons were co-immunostained for LC3B and BAG3, SYNPO or SQSTM1, respectively. Overlap of BAG3, SYNPO or SQSTM1 with LC3B puncta was observed in neuronal processes. SYN1 was used as a negative control. The corresponding line scans are shown at right. Arrowheads denote areas of overlap. Scale bar: 10 μm; scale bar in the high magnification inserts: 2 μm. ( B ) Quantification of colocalization using Pearson’s correlation coefficient. ( C ) Quantification of colocalization using object based analysis. In each condition, 12-20 neurons from 3 independent experiments were used for quantification. Graphs were plotted as mean ± SEM. Colocalization between LC3B and SYNPO, BAG3 and SQSTM1, respectively, was compared to LC3B and SYN1 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p
Figure Legend Snippet: Colocalization of BAG3, SYNPO or SQSTM1 with endogenous MAP1LC3B/LC3B in neuronal processes. ( A ) Neurons were co-immunostained for LC3B and BAG3, SYNPO or SQSTM1, respectively. Overlap of BAG3, SYNPO or SQSTM1 with LC3B puncta was observed in neuronal processes. SYN1 was used as a negative control. The corresponding line scans are shown at right. Arrowheads denote areas of overlap. Scale bar: 10 μm; scale bar in the high magnification inserts: 2 μm. ( B ) Quantification of colocalization using Pearson’s correlation coefficient. ( C ) Quantification of colocalization using object based analysis. In each condition, 12-20 neurons from 3 independent experiments were used for quantification. Graphs were plotted as mean ± SEM. Colocalization between LC3B and SYNPO, BAG3 and SQSTM1, respectively, was compared to LC3B and SYN1 using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****, p

Techniques Used: Negative Control

The BAG3 WW domain and SYNPO PPxY motifs are required for their interaction. ( A ) BAG3 is a multi-domain protein, which contains a WW domain at its amino-terminus for binding PPxY motifs in partner proteins. In a previously performed peptide array screen for BAG3 WW domain interacting proteins of the human proteome [ 18 ], 12-mer peptides of SYNPO2 (aa 615-626) and SYNPO (aa 333-344), respectively, were strongly recognized by the WW domain of the co-chaperone BAG3. ( B ) HeLa cells were transiently transfected with empty plasmid or plasmid constructs for the expression of FLAG-tagged SYNPO or mutant forms with inactivating mutations in the PPxY motifs, as indicated followed by immunoprecipitation with an anti-FLAG antibody (IP). Isolated immune complexes were probed for the presence of endogenous BAG3. Input samples correspond to 32 μg of protein. ( C ) Similar to the experimental approach described under ( B ), BAG3 complexes were isolated from HeLa cells expressing a wild-type form of the BAG3 co-chaperone or a form with an inactivated WW domain (BAG3-WAWA). Isolated complexes were analyzed for the presence of endogenous SYNPO.
Figure Legend Snippet: The BAG3 WW domain and SYNPO PPxY motifs are required for their interaction. ( A ) BAG3 is a multi-domain protein, which contains a WW domain at its amino-terminus for binding PPxY motifs in partner proteins. In a previously performed peptide array screen for BAG3 WW domain interacting proteins of the human proteome [ 18 ], 12-mer peptides of SYNPO2 (aa 615-626) and SYNPO (aa 333-344), respectively, were strongly recognized by the WW domain of the co-chaperone BAG3. ( B ) HeLa cells were transiently transfected with empty plasmid or plasmid constructs for the expression of FLAG-tagged SYNPO or mutant forms with inactivating mutations in the PPxY motifs, as indicated followed by immunoprecipitation with an anti-FLAG antibody (IP). Isolated immune complexes were probed for the presence of endogenous BAG3. Input samples correspond to 32 μg of protein. ( C ) Similar to the experimental approach described under ( B ), BAG3 complexes were isolated from HeLa cells expressing a wild-type form of the BAG3 co-chaperone or a form with an inactivated WW domain (BAG3-WAWA). Isolated complexes were analyzed for the presence of endogenous SYNPO.

Techniques Used: Binding Assay, Peptide Microarray, Transfection, Plasmid Preparation, Construct, Expressing, Mutagenesis, Immunoprecipitation, Isolation

SYNPO does not show significant overlap with MAP2. Immunofluorescence images of mature neurons stained for SYNPO (green) and MAP2 (red). Merged images shows that SYNPO shows little overlap with MAP2 in neuronal processes (images in the bottom row). Corresponding line scanning of a process is shown at the right. Scale bar: 10 μm.
Figure Legend Snippet: SYNPO does not show significant overlap with MAP2. Immunofluorescence images of mature neurons stained for SYNPO (green) and MAP2 (red). Merged images shows that SYNPO shows little overlap with MAP2 in neuronal processes (images in the bottom row). Corresponding line scanning of a process is shown at the right. Scale bar: 10 μm.

Techniques Used: Immunofluorescence, Staining

SYNPO or BAG3 knockdown does not affect lysosomal function. ( A ) Maturation of CTSL (cathepsin L) in either BAG3 or SYNPO knockdown neurons by immunoblotting. pro., precursor CTSL; im, immature CTSL; m, mature CTSL. Neurons treated with 10 μM chloroquine (CQ) were used as a positive control. ( B ) Quantification of precursor CTSL:mature CTSL ratio. Graph shows mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p
Figure Legend Snippet: SYNPO or BAG3 knockdown does not affect lysosomal function. ( A ) Maturation of CTSL (cathepsin L) in either BAG3 or SYNPO knockdown neurons by immunoblotting. pro., precursor CTSL; im, immature CTSL; m, mature CTSL. Neurons treated with 10 μM chloroquine (CQ) were used as a positive control. ( B ) Quantification of precursor CTSL:mature CTSL ratio. Graph shows mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. **, p

Techniques Used: Positive Control

BAG3 or SYNPO knockdown blocks the autophagic flux of autophagy in neuronal processes. Representative maximal-projections of confocal z-stack images of neuronal soma ( A ) and processes ( B ). Neurons treated with 100 nM bafilomycin A1 (BafA1) for 4 h were used as positive controls. Scale bar: 10 μm. ( C ) Quantification of autophagosomes (green) and autolysosomes (red only) under the conditions of ( A ) and ( B ). The total number of green particles (autophagosomes) and red particles (autophagosomes plus autolysosomes) were counted as described in Materials and Methods. Red only particles (autolysosomes) were determined by subtracting the number of green particles from the respective number of red particles. Data were obtained from 20-30 neurons of 3 independent experiments. One to three processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Dunnett’s post hoc test. *, p
Figure Legend Snippet: BAG3 or SYNPO knockdown blocks the autophagic flux of autophagy in neuronal processes. Representative maximal-projections of confocal z-stack images of neuronal soma ( A ) and processes ( B ). Neurons treated with 100 nM bafilomycin A1 (BafA1) for 4 h were used as positive controls. Scale bar: 10 μm. ( C ) Quantification of autophagosomes (green) and autolysosomes (red only) under the conditions of ( A ) and ( B ). The total number of green particles (autophagosomes) and red particles (autophagosomes plus autolysosomes) were counted as described in Materials and Methods. Red only particles (autolysosomes) were determined by subtracting the number of green particles from the respective number of red particles. Data were obtained from 20-30 neurons of 3 independent experiments. One to three processes from each neuron were chosen for analysis. Data were shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Dunnett’s post hoc test. *, p

Techniques Used:

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    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
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    Santa Cruz Biotechnology synaptopodin
    Lithium counteracts the Adriamycin (ADR; doxorubicin)-induced GSK3β overactivity and paxillin hyperphosphorylation in glomeruli and reinstates actin cytoskeleton integrity in glomerular podocytes. A: Glomeruli were isolated from kidneys from differently treated animals by the magnetic beads–based approach and were homogenized for immunoblot analysis for phosphorylated GSK3β, phosphorylated paxillin, total GSK3β, total paxillin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Densitometric Western blot analysis estimates the relative levels of phosphorylated GSK3β/total GSK3β ratios and phosphorylated paxillin/total paxillin ratios in isolated glomeruli from different groups. C: Frozen kidney sections procured on day 14 were subjected to phalloidin labeling of F-actin (red) as well as immunofluorescence staining for <t>synaptopodin</t> (green), a podocyte marker. Confocal microscopy images. ADR injury not only reduces synaptopodin expression but also diminishes the integrated pixel density of the merged areas (yellow), where F-actin co-localizes with synaptopodin, suggesting a disorganized actin cytoskeletal network in the remnant intact podocytes. Computerized morphometric analysis of the ratios of integrated pixel densities between yellow signal to green signal in immunofluorescence micrographs obtained in C and D . Data are given as means ± SD. n = 6 ( B and D ). ∗ P
    Synaptopodin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Lithium counteracts the Adriamycin (ADR; doxorubicin)-induced GSK3β overactivity and paxillin hyperphosphorylation in glomeruli and reinstates actin cytoskeleton integrity in glomerular podocytes. A: Glomeruli were isolated from kidneys from differently treated animals by the magnetic beads–based approach and were homogenized for immunoblot analysis for phosphorylated GSK3β, phosphorylated paxillin, total GSK3β, total paxillin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Densitometric Western blot analysis estimates the relative levels of phosphorylated GSK3β/total GSK3β ratios and phosphorylated paxillin/total paxillin ratios in isolated glomeruli from different groups. C: Frozen kidney sections procured on day 14 were subjected to phalloidin labeling of F-actin (red) as well as immunofluorescence staining for synaptopodin (green), a podocyte marker. Confocal microscopy images. ADR injury not only reduces synaptopodin expression but also diminishes the integrated pixel density of the merged areas (yellow), where F-actin co-localizes with synaptopodin, suggesting a disorganized actin cytoskeletal network in the remnant intact podocytes. Computerized morphometric analysis of the ratios of integrated pixel densities between yellow signal to green signal in immunofluorescence micrographs obtained in C and D . Data are given as means ± SD. n = 6 ( B and D ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Glycogen Synthase Kinase 3β Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity

    doi: 10.1016/j.ajpath.2014.06.027

    Figure Lengend Snippet: Lithium counteracts the Adriamycin (ADR; doxorubicin)-induced GSK3β overactivity and paxillin hyperphosphorylation in glomeruli and reinstates actin cytoskeleton integrity in glomerular podocytes. A: Glomeruli were isolated from kidneys from differently treated animals by the magnetic beads–based approach and were homogenized for immunoblot analysis for phosphorylated GSK3β, phosphorylated paxillin, total GSK3β, total paxillin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Densitometric Western blot analysis estimates the relative levels of phosphorylated GSK3β/total GSK3β ratios and phosphorylated paxillin/total paxillin ratios in isolated glomeruli from different groups. C: Frozen kidney sections procured on day 14 were subjected to phalloidin labeling of F-actin (red) as well as immunofluorescence staining for synaptopodin (green), a podocyte marker. Confocal microscopy images. ADR injury not only reduces synaptopodin expression but also diminishes the integrated pixel density of the merged areas (yellow), where F-actin co-localizes with synaptopodin, suggesting a disorganized actin cytoskeletal network in the remnant intact podocytes. Computerized morphometric analysis of the ratios of integrated pixel densities between yellow signal to green signal in immunofluorescence micrographs obtained in C and D . Data are given as means ± SD. n = 6 ( B and D ). ∗ P

    Article Snippet: The antibodies against paxillin, GSK3β, p-GSK3β (S9), synaptopodin, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and those against paxillin, phosphorylated paxillin (S126), and nephrin were acquired from Cell Signaling Technology Inc. (Danvers, MA) and Progen Biotechnik GmbH (Heidelberg, Germany), respectively.

    Techniques: Isolation, Magnetic Beads, Western Blot, Labeling, Immunofluorescence, Staining, Marker, Confocal Microscopy, Expressing

    Lithium corrects the Adriamycin (ADR; doxorubicin)-accelerated dynamics of FA turnover in podocytes. A: Differentiated podocytes were transiently transfected with a vector encoding green fluorescent protein (GFP)–paxillin and were subjected to time-lapse microscopy for 1 hour, followed by fluorescence immunocytochemical staining for synaptopodin. Representative fluorescent micrographs of synaptopodin staining showed that podocytes retain the podocyte marker protein synaptopodin. B: Podocytes transfected with GFP-paxillin were injured with ADR for 8 hours after 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl) treatment for 6 hours. Subsequently, live podocytes were subjected to time-lapse fluorescence microscopy for 1 hour with 2 minutes between microscopic image frames. The Detail column represents a series of time-lapse microscopic image frames aligned to show the temporal evolution of individual FAs from assembly to disassembly in differently treated podocytes. Because image frames were captured at a fixed rate (0.5 frames per minute), the number of image frames showing the temporal evolution of an individual FA accordingly correlated the FA dynamics. Thus, hypodynamics and hyperdynamics of FA turnover were indicated by more and less frames, respectively. ADR-treated podocytes shrank rapidly, as shown by the whole cell image and exhibit an accelerated FA turnover (Detail column). This effect was abrogated by lithium pretreatment. Boxed regions focal adhesions, whose turnover is shown in Detail column. C: Quantification of FA assembly rates. Lithium treatment alone slightly reduced the assembly rate. ADR drastically increased the FA assembly rate, which was significantly obliterated by lithium pretreatment. D: Quantification of FA disassembly rates. ADR markedly increased the FA disassembly rate, and lithium pretreatment prevented the effect. Horizontal bars indicate the median values and the top and bottom lines of the boxes indicate the 3rd and 1st quartile, respectively ( C and D ). Data are given as medians ± ranges ( C and D ). n = 30 cells from six experiments ( C and D ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Glycogen Synthase Kinase 3β Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity

    doi: 10.1016/j.ajpath.2014.06.027

    Figure Lengend Snippet: Lithium corrects the Adriamycin (ADR; doxorubicin)-accelerated dynamics of FA turnover in podocytes. A: Differentiated podocytes were transiently transfected with a vector encoding green fluorescent protein (GFP)–paxillin and were subjected to time-lapse microscopy for 1 hour, followed by fluorescence immunocytochemical staining for synaptopodin. Representative fluorescent micrographs of synaptopodin staining showed that podocytes retain the podocyte marker protein synaptopodin. B: Podocytes transfected with GFP-paxillin were injured with ADR for 8 hours after 10 mmol/L lithium chloride (LiCl) or 10 mmol/L sodium chloride (NaCl) treatment for 6 hours. Subsequently, live podocytes were subjected to time-lapse fluorescence microscopy for 1 hour with 2 minutes between microscopic image frames. The Detail column represents a series of time-lapse microscopic image frames aligned to show the temporal evolution of individual FAs from assembly to disassembly in differently treated podocytes. Because image frames were captured at a fixed rate (0.5 frames per minute), the number of image frames showing the temporal evolution of an individual FA accordingly correlated the FA dynamics. Thus, hypodynamics and hyperdynamics of FA turnover were indicated by more and less frames, respectively. ADR-treated podocytes shrank rapidly, as shown by the whole cell image and exhibit an accelerated FA turnover (Detail column). This effect was abrogated by lithium pretreatment. Boxed regions focal adhesions, whose turnover is shown in Detail column. C: Quantification of FA assembly rates. Lithium treatment alone slightly reduced the assembly rate. ADR drastically increased the FA assembly rate, which was significantly obliterated by lithium pretreatment. D: Quantification of FA disassembly rates. ADR markedly increased the FA disassembly rate, and lithium pretreatment prevented the effect. Horizontal bars indicate the median values and the top and bottom lines of the boxes indicate the 3rd and 1st quartile, respectively ( C and D ). Data are given as medians ± ranges ( C and D ). n = 30 cells from six experiments ( C and D ). ∗ P

    Article Snippet: The antibodies against paxillin, GSK3β, p-GSK3β (S9), synaptopodin, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and those against paxillin, phosphorylated paxillin (S126), and nephrin were acquired from Cell Signaling Technology Inc. (Danvers, MA) and Progen Biotechnik GmbH (Heidelberg, Germany), respectively.

    Techniques: Transfection, Plasmid Preparation, Time-lapse Microscopy, Fluorescence, Staining, Marker, Microscopy

    miR-146a levels are reduced in the diabetic human and mouse kidney glomeruli. A and B, representative ISH images of human ( A ) and mouse ( B ) kidney sections to detect the expression pattern of miR-146a (indicated with an arrow ). Each kidney section was stained with the indicated probe (against miR-146a, a scrambled control or against U6 RNA). Confocal images of immunofluorescently labeled glomeruli ( A ), stained with the podocyte marker synaptopodin (Synpo, green ), show relative podocyte density in the representative healthy and diabetic human kidney sections. Representative ISH images of kidney sections from C57BL/6 WT and miR-146a −/− animals stained with a specific probe against miR-146a are also shown ( B ). Scale bar , 50 μm ( A and B ). C, a bar graph showing urinary ACR in 12-week-old BTBR WT and BTBR ob / ob animals. Data shown are mean ± S.E. ( n = 5/group). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1 *

    doi: 10.1074/jbc.M116.753822

    Figure Lengend Snippet: miR-146a levels are reduced in the diabetic human and mouse kidney glomeruli. A and B, representative ISH images of human ( A ) and mouse ( B ) kidney sections to detect the expression pattern of miR-146a (indicated with an arrow ). Each kidney section was stained with the indicated probe (against miR-146a, a scrambled control or against U6 RNA). Confocal images of immunofluorescently labeled glomeruli ( A ), stained with the podocyte marker synaptopodin (Synpo, green ), show relative podocyte density in the representative healthy and diabetic human kidney sections. Representative ISH images of kidney sections from C57BL/6 WT and miR-146a −/− animals stained with a specific probe against miR-146a are also shown ( B ). Scale bar , 50 μm ( A and B ). C, a bar graph showing urinary ACR in 12-week-old BTBR WT and BTBR ob / ob animals. Data shown are mean ± S.E. ( n = 5/group). ***, p

    Article Snippet: Sections were blocked at room temperature for 1 h and incubated with the primary antibodies against total Notch-1 (polyclonal rabbit anti-Notch1, (catalog number 100-401-407, Rockland, Limerick, PA), ErbB4 (polyclonal rabbit anti-ErBB4, catalog number sc-283, C-18, Santa Cruz, Dallas, TX), EGFR (polyclonal rabbit anti-EGFR catalog number 06-847, Millipore, Darmstadt, Germany), and synaptopodin (polyclonal goat, SC-21537, Santa Cruz) at 4 °C overnight.

    Techniques: In Situ Hybridization, Expressing, Staining, Labeling, Marker

    STZ treatment of WT and miR-146a −/− mice results in increased glomerular injury and induction of miR-146a targets in the mouse glomeruli that is suppressed by erlotinib. A, STZ-induced up-regulation of EGFR, Notch-1, and ErbB4 expression in the mouse glomeruli is reduced by erlotinib. Representative confocal microscopy images of immunofluorescently labeled glomeruli from WT ( top panels ) and miR-146a −/− ( bottom panels ) mice treated with vehicle alone ( Control ), with STZ and vehicle ( STZ ) or with STZ and erlotinib ( STZ + Erl ). Kidney sections were imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4, and Synaptopodin ( Synpo ). Merged DAPI, EGFR and Synpo, DAPI, Notch-1 and Synpo, and DAPI, ErbB4 and Synpo channels are also presented that show podocyte colocalization for these proteins. Scale bar , 50 μm. B , graphs showing the quantification of relative glomerular signal intensity of EGFR, Notch-1, and ErbB4 in samples from A . Data shown are mean ± S.E. ( n = 5/group). ns , not significant; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1 *

    doi: 10.1074/jbc.M116.753822

    Figure Lengend Snippet: STZ treatment of WT and miR-146a −/− mice results in increased glomerular injury and induction of miR-146a targets in the mouse glomeruli that is suppressed by erlotinib. A, STZ-induced up-regulation of EGFR, Notch-1, and ErbB4 expression in the mouse glomeruli is reduced by erlotinib. Representative confocal microscopy images of immunofluorescently labeled glomeruli from WT ( top panels ) and miR-146a −/− ( bottom panels ) mice treated with vehicle alone ( Control ), with STZ and vehicle ( STZ ) or with STZ and erlotinib ( STZ + Erl ). Kidney sections were imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4, and Synaptopodin ( Synpo ). Merged DAPI, EGFR and Synpo, DAPI, Notch-1 and Synpo, and DAPI, ErbB4 and Synpo channels are also presented that show podocyte colocalization for these proteins. Scale bar , 50 μm. B , graphs showing the quantification of relative glomerular signal intensity of EGFR, Notch-1, and ErbB4 in samples from A . Data shown are mean ± S.E. ( n = 5/group). ns , not significant; **, p

    Article Snippet: Sections were blocked at room temperature for 1 h and incubated with the primary antibodies against total Notch-1 (polyclonal rabbit anti-Notch1, (catalog number 100-401-407, Rockland, Limerick, PA), ErbB4 (polyclonal rabbit anti-ErBB4, catalog number sc-283, C-18, Santa Cruz, Dallas, TX), EGFR (polyclonal rabbit anti-EGFR catalog number 06-847, Millipore, Darmstadt, Germany), and synaptopodin (polyclonal goat, SC-21537, Santa Cruz) at 4 °C overnight.

    Techniques: Mouse Assay, Expressing, Confocal Microscopy, Labeling, Staining

    Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P

    Journal: PLoS ONE

    Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

    doi: 10.1371/journal.pone.0063799

    Figure Lengend Snippet: Aggravation of podocyte injury upon autophagy inhibition. (A–B).The Western blot results confirmed that the expression of synaptopodin and LC3 II was lower in ATG7 (a key protein of autophagy) RNAi-mediated knock down differentiated MPCs (siRNA+R+P) than in the siRNA negative control group (siCon+R+P). Both groups were pretreated with rapamycin and treated with PAN,* P

    Article Snippet: The following primary antibodies were used: rabbit anti-synaptopodin and goat anti-synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p-mTOR (Ser2448) (Cell Signaling, Danvers, MA); anti-p-70S6K (Thr389) (Cell Signaling); anti-p-4EBP1 (Ser65) (Cell Signaling); anti-p-ULK1 (Ser757) (Cell Signaling); rabbit anti-LC3 (Sigma-Aldrich) and moduse anti-LC3 (MBL Co. NaKa-ku Nagoya, Japan); and mouse anti-β-actin (Sigma-Aldrich).

    Techniques: Inhibition, Western Blot, Expressing, Negative Control

    Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P

    Journal: PLoS ONE

    Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

    doi: 10.1371/journal.pone.0063799

    Figure Lengend Snippet: Rapamycin reduced podocyte injury by inhibiting the mTOR-ULK1 signaling pathway. (A–B).Western blot analysis of cellular proteins showed recovery of synaptopodin and LC3 II expression, a decrease in mTOR activity, and a decrease in ULK1 phosphorylation in rapamycin-pretreated cells (P+R) compared to PAN-treated cells in the absence of rapamycin (PAN), * P

    Article Snippet: The following primary antibodies were used: rabbit anti-synaptopodin and goat anti-synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p-mTOR (Ser2448) (Cell Signaling, Danvers, MA); anti-p-70S6K (Thr389) (Cell Signaling); anti-p-4EBP1 (Ser65) (Cell Signaling); anti-p-ULK1 (Ser757) (Cell Signaling); rabbit anti-LC3 (Sigma-Aldrich) and moduse anti-LC3 (MBL Co. NaKa-ku Nagoya, Japan); and mouse anti-β-actin (Sigma-Aldrich).

    Techniques: Western Blot, Expressing, Activity Assay

    Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P

    Journal: PLoS ONE

    Article Title: Rapamycin Upregulates Autophagy by Inhibiting the mTOR-ULK1 Pathway, Resulting in Reduced Podocyte Injury

    doi: 10.1371/journal.pone.0063799

    Figure Lengend Snippet: Preparation of passive Heymann nephritis rat model. (A).At day 1, 7, 14, 21, and 28 after antiserum injection, rats were sacrificed and the renal cortex was removed. Cryosections of the renal cortex were stained with goat anti-rat IgG-fluorescein isothiocyanate (FITC). Microscopic examination revealed that there were IgG depositions in the glomeruli of PHN rats compared to those of control rats. Depositions gradually increased over time, peaking on day 14. Magnification = × 400 (B). 24-hr urine was collected on days 0 (control), 1, 7, 14, 21, and 28, and analyzed for urinary albumin content using Coomassie Brilliant Blue G-250. (C). Electrophoresis followed by Coomassie blue staining. Each group urine sample was subjected to SDS-PAGE. Obvious albuminuria was apparent from day 7. (D-E).Western blot analysis of isolated glomerular protein on day 14 showed significantly reduced expression of synaptopodin in PHN rats compared to that in controls, * P

    Article Snippet: The following primary antibodies were used: rabbit anti-synaptopodin and goat anti-synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p-mTOR (Ser2448) (Cell Signaling, Danvers, MA); anti-p-70S6K (Thr389) (Cell Signaling); anti-p-4EBP1 (Ser65) (Cell Signaling); anti-p-ULK1 (Ser757) (Cell Signaling); rabbit anti-LC3 (Sigma-Aldrich) and moduse anti-LC3 (MBL Co. NaKa-ku Nagoya, Japan); and mouse anti-β-actin (Sigma-Aldrich).

    Techniques: Injection, Staining, Electrophoresis, SDS Page, Western Blot, Isolation, Expressing