Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

doi: 10.4049/jimmunol.2100483

Figure Lengend Snippet: FIGURE 1. AIM2 expression in nor- mal human kidney. (A) Immunoblot probing for AIM2 in two independent human kidney samples using Cell Sig- naling Technology and Sigma-Aldrich Abs. THP-1 cells plus or minus g-IFN are used as controls. Arrows denote AIM2 forms. (B) AIM2 mRNA expres- sion in glomerular fraction normalized to total human kidney by qPCR. Glomeruli were isolated from human kidney tissue by a sieving technique. Mean ± SD, n 5 4. *p < 0.05, Student t test. A represen- tative image of a PAS-stained normal human glomerulus is shown in the lower panel (original magnification 40). (C) Indirect immunofluorescence confocal microscopy probing for AIM2 (red) and WT1, synaptopodin, PDGFRb, and CD31 (green) in paraffin-embedded nor- mal kidney tissue. Nuclear staining using DAPI is shown in merged images (blue). Dashed circle (G) indicates glomeruli in the kidney. Right panel represents mag- nified view of hatched inset. Dual- labeling confocal microscopy shows localization of AIM2 to podocytes. Scale bars, 50 mm. Images are representative of staining performed at least three inde- pendent times. (D) RNA in situ hybridi- zation (RNAScope) probing for AIM2, NHPS1 (nephrin), and CD44 in normal human glomerulus. AIM2 mRNA coloc- alizes with NHPS1 but not CD44 in the normal kidney. (iiii) Magnified views of indicated hatched boxes. Scale bar, 25 mm. Images are representative of experiments performed at least three independent times.

Article Snippet: The following Abs were used: anti-human AIM2 (polyclonal; SigmaAldrich), anti-human AIM2 (D5X7K; Cell Signaling Technology), antihuman AIM2 (affinity-purified) (41), anti-mouse AIM2 (polyclonal; Cell Signaling Technology), anti-human PDGF-receptor-b (polyclonal; Abcam), anti-human synaptopodin (918841; R&D Systems), anti-human CD31 (9G11; R&D Systems), anti-human WT1 (6F-H2; EMD Millipore), antihuman WT1 (F-6; Santa Cruz Biotechnology), anti-mouse WT1 (polyclonal; Santa Cruz Biotechnology), anti-human pro IL-1b (2805; R&D Systems), anti-human caspase-1 (polyclonal; Santa Cruz Biotechnology), anti-human gasdermin D (polyclonal; Sigma-Aldrich), b-tubulin (AA2; Sigma-Aldrich), and anti-human CD44 (G44-26; BD Biosciences).

Techniques: Expressing, Western Blot, Isolation, Staining, Immunofluorescence, Confocal Microscopy, Labeling, In Situ, RNAscope

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

doi: 10.4049/jimmunol.2100483

Figure Lengend Snippet: FIGURE 2. AIM2 and inflammasome activation in primary human podo- cytes. (A) Immunoblotting for AIM2 and synaptopodin in primary human podocytes. Human kidney and THP-1 cells plus or minus g-IFN stimulation are used as controls. Arrows denote AIM2 forms. (B) ELISA for IL-1b in supernatant of LPS-primed or -unprimed podocytes treated with nigericin (50 mM, 1 h) or transfected with poly(dA:dT) (2 mg/ml, 3 h). PMA-primed THP-1 cells treated with poly(dA:dT) (2 mg/ml, 3 h) were used as a posi- tive control. (C) Immunoblotting probing for caspase-1 (p45 proform and p11, cleaved CARD fragment), proIL-1b and gasdermin D (GSDMD) in unprimed or LPS-primed human podocytes. PMA-primed THP-1 cells treated with nigericin were used as a positive control for inflammasome activation. (D) ELISA for IL-6 secretion in unprimed or LPS-primed human podocytes. PMA-primed THP-1 cells are used as a positive control. Repre- sentative of experiments were performed at least three independent times. nd, not detected.

Article Snippet: The following Abs were used: anti-human AIM2 (polyclonal; SigmaAldrich), anti-human AIM2 (D5X7K; Cell Signaling Technology), antihuman AIM2 (affinity-purified) (41), anti-mouse AIM2 (polyclonal; Cell Signaling Technology), anti-human PDGF-receptor-b (polyclonal; Abcam), anti-human synaptopodin (918841; R&D Systems), anti-human CD31 (9G11; R&D Systems), anti-human WT1 (6F-H2; EMD Millipore), antihuman WT1 (F-6; Santa Cruz Biotechnology), anti-mouse WT1 (polyclonal; Santa Cruz Biotechnology), anti-human pro IL-1b (2805; R&D Systems), anti-human caspase-1 (polyclonal; Santa Cruz Biotechnology), anti-human gasdermin D (polyclonal; Sigma-Aldrich), b-tubulin (AA2; Sigma-Aldrich), and anti-human CD44 (G44-26; BD Biosciences).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Positive Control

Journal: Journal of the American Society of Nephrology

Article Title: Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function

doi: 10.1681/asn.2015010032

Figure Lengend Snippet: Figure 4. p18 in podocytes is dispensable for glomerular function. (A) Representative periodic acid–Schiff (PAS) staining and TEM images of the glomeruli of age-matched WT and podo-p18 KO mice are shown. (B) Immunohistochemistry analysis for nephrin and synaptopodin localization in the glomeruli of WT and podo-p18 KO mice. (C) 24-hour urine was collected from WT and podo-p18 KO mice at the indicated time points (n.4 for each genotype in each time point) and albumin and creatinine concentrations were measured. The results are shown as ratios of micrograms of albumin by milligrams of creatinine.

Article Snippet: Antibodies were used at the following dilutions: pS6 (1:300; Cell Signaling Technology); pAkt S473 (1:400; Cell Signaling Technology); synaptopodin (1:100; Genway); col- lagen type IV (1:150; EMD Millipore, Billerica, MA);fibronectin (1:400; Sigma-Aldrich),Desmin (1:50; Dako), Bip (1:100; Abcam, Inc.).

Techniques: Staining, Immunohistochemistry

Journal: Journal of the American Society of Nephrology

Article Title: Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function

doi: 10.1681/asn.2015010032

Figure Lengend Snippet: Figure 6. Ablation of p18 in the podocytes prevents hyper-activation of mTORC1 and podocyte injury in podo-TSC1 KO mice. (A) Immunohistochemistry analysis of mTORC1 activity and Akt activity in the glomeruli from 8-week-old WT, podo-TSC1 KO, and podo-TSC1/p18 DKO mice. (B) Glomeruli were isolated from mice of the above genetic backgrounds. Tissue lysates were analyzed by Western blotting to examine mTORC1 activity and Akt phosphorylation. (C) Renal tissues from the indicated animals were double stained with Bip and synaptopodin, or Desmin and WT1. (D) Immunohistochemistry analysis of nephrin and synaptopodin localization in the glomeruli of the indicated mice. Areas indicated by squares in the Merge column were shown at higher magnification with individual channels. (E) Kidney sections from WT, podo-TSC1 KO, and podo-TSC1/p18 DKO mice were stained with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), fibronectin (FN), and type IV collagen (COL4). Glomerular tuft area and PAS-positive staining areas within the glomeruli of the indicated mice were quantified. For each genotype, 20 pictures were taken of different fields and used for quantification. *P,0.01 versus other groups; mean6SEM (n= approximately 3–4 mice).

Article Snippet: Antibodies were used at the following dilutions: pS6 (1:300; Cell Signaling Technology); pAkt S473 (1:400; Cell Signaling Technology); synaptopodin (1:100; Genway); col- lagen type IV (1:150; EMD Millipore, Billerica, MA);fibronectin (1:400; Sigma-Aldrich),Desmin (1:50; Dako), Bip (1:100; Abcam, Inc.).

Techniques: Activation Assay, Immunohistochemistry, Activity Assay, Isolation, Western Blot, Phospho-proteomics, Staining

Journal: Journal of the American Society of Nephrology

Article Title: Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function

doi: 10.1681/asn.2015010032

Figure Lengend Snippet: Figure 8. Ablation of p18 in the podocytes prevents mesangial expansion in diabetic mice. (A) Kidney sections from the indicated 20- week-old mice were stained with pS6, synaptopodin, periodic acid–Schiff (PAS), type IV collagen, and fibronectin. (B–D) Quantifications of PAS-, type IV collagen-, and fibronectin-positive area within a glomerulus were shown. Ratios (positive area/ glomerular tuft area) were determined in 40 glomeruli from the indicated mice and expressed as the mean fold change. **P,0.001; mean6SEM (n=4 mice).

Article Snippet: Antibodies were used at the following dilutions: pS6 (1:300; Cell Signaling Technology); pAkt S473 (1:400; Cell Signaling Technology); synaptopodin (1:100; Genway); col- lagen type IV (1:150; EMD Millipore, Billerica, MA);fibronectin (1:400; Sigma-Aldrich),Desmin (1:50; Dako), Bip (1:100; Abcam, Inc.).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

doi: 10.1083/jcb.201412003

Figure Lengend Snippet: Mechanical force induces α-actinin-4 and actin accumulation at the cell junction. (A) Deconvolved optical section at the apical junction showing tension-induced α-actinin-4 and actin accumulation in maturing monolayers. (B) Quantitation of junctional stainings in A before (U) and after (P) cyclic pressure showing sixfold induction of α-actinin-4 and twofold induction of actin accumulation. (C) Deconvolved optical section and z-composites at the apical junction showing tension-induced α-actinin-4 and actin accumulation in mature monolayers. (D) Quantitation of junctional stainings in C before (U) and after (P) cyclic pressure showing a modest increase in α-actinin-4 and actin accumulation. (E) Deconvolved optical section at the apical junction showing tension-induced α-actinin-4 accumulation by prolonged cyclic basal 2 mmHg pressure despite decrease in β-catenin and p120 (pink arrowheads). (F) Quantitation of junctional stainings in E before (U) and after (P) cyclic pressure. (G) Normalization of junctional staining in E showing sixfold induction of α-actinin-4. (H) Deconvolved optical section at the apical junction showing unchanged levels of synaptopodin after prolonged cyclic pressure. White arrowheads show colocalization of synaptopodin, α-actinin-4, and β-catenin. (I) Quantitation of junctional stainings in H before (U) and after (P) cyclic pressure. (J) Normalization of junctional staining in H showing sixfold induction of α-actinin-4. (B, D, F, G, I, and J) Means are represented by horizontal lines. Bars, 5 µm.

Article Snippet: The coding sequence of human synaptopodin cDNAs (OriGene catalog no. RC228616) corresponding to aa 1–929, aa 1–600, aa 245–600, and aa 600–929 were subcloned into Sal1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with CBP and SBP tags at the N terminus for expression in MDCK cells. shRNAs for canine α-actinin-4 (5′-AGGTCCTGTTCCTCTGACTCGGTATCTAT-3′) and canine synaptopodin (5′-GAGGTGAGATCCAGCACACTTCTGATTGA-3′) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS and puromycin-selectable pRS vectors, respectively (OriGene).

Techniques: Quantitation Assay, Staining

Journal: The Journal of Cell Biology

Article Title: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

doi: 10.1083/jcb.201412003

Figure Lengend Snippet: Identification of synaptopodin as a mechanosensitive junctional protein. (A) Peptides (green font) from α-actinin-4 cross-linking experiment matching synaptopodin sequence. Basic residues lysine (K) and arginine (R) are underlined. (B) Deconvolved optical section at the apical junction showing colocalization of synaptopodin, α-actinin-4, E-cadherin, and β-catenin in mature monolayer. Yellow arrowheads show colocalization of synaptopodin, α-actinin-4, and E-cadherin. (C) Deconvolved optical section at the apical junction showing colocalization of synaptopodin, α-actinin-4, E-cadherin, and actin (phalloidin) at latrunculin-resistant junctional puncta (orange arrowheads). (D) Deconvolved optical section at the apical junction showing tension-induced junctional accumulation of synaptopodin and α-actinin-4 (orange arrowheads). (E) Quantitation of junctional synaptopodin, α-actinin-4, and actin before (U) and after (P) cyclic basal 2 mmHg pressure in young monolayers. Means are represented by horizontal lines. (F) Correlation of junctional α-actinin-4 and E-cadherin before (No P) and after (Low P) cyclic basal 2 mmHg pressure in young monolayers. (G) Correlation of synaptopodin and α-actinin-4 junctional levels before (No Pressure) and after (Low Pressure) cyclic basal 2 mmHg pressure in young monolayers. (H) Correlation of synaptopodin and actin junctional levels before (No Pressure) and after (Low Pressure) cyclic basal 2 mmHg pressure in young monolayers. (I) Correlation of α-actinin-4 and actin junctional levels before (No Pressure) and after (Low Pressure) cyclic basal 2 mmHg pressure in young monolayers. (D–I) The images and quantitation shown are from a single representative experiment out of six experiments. Bars, 2 µm.

Article Snippet: The coding sequence of human synaptopodin cDNAs (OriGene catalog no. RC228616) corresponding to aa 1–929, aa 1–600, aa 245–600, and aa 600–929 were subcloned into Sal1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with CBP and SBP tags at the N terminus for expression in MDCK cells. shRNAs for canine α-actinin-4 (5′-AGGTCCTGTTCCTCTGACTCGGTATCTAT-3′) and canine synaptopodin (5′-GAGGTGAGATCCAGCACACTTCTGATTGA-3′) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS and puromycin-selectable pRS vectors, respectively (OriGene).

Techniques: Sequencing, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

doi: 10.1083/jcb.201412003

Figure Lengend Snippet: Synaptopodin is required for α-actinin-4 recruitment to the cell junction. (A) Deconvolved optical section at the apical junction showing unchanged levels of E-cadherin, synaptopodin, and α-actinin-4 after prolonged cyclic pressure in mature cell monolayer. (B) Quantitation of junctional stainings in A before (U) and after (P) cyclic pressure. (C) Western blots showing upward shift of synaptopodin (black arrowheads) in a tension-sensitive manner. (D) Deconvolved optical section at the apical junction showing decreased α-actinin-4 and actin accumulation at the cell junction in synaptopodin knockdown cells. (E) Quantification of D before (U) and after (P) cyclic pressure. (B and E) Means are represented by horizontal lines. (F) Western blots showing α-actinin-4 level unchanged by knockdown of synaptopodin. (G) Wide-field images of venus-actinin showing cytoplasmic localization in synaptopodin knockdown cells. (H) BSA flux assays showing decreased barrier formation in synaptopodin knockdown monolayers. (I) Wide-field images showing synaptopodin knockdown cells detaching from monolayer upon mechanical insult. (J) Western blots showing synaptopodin level unchanged by knockdown of α-actinin-4. (K) Deconvolved optical section showing normal synaptopodin colocalization with E-cadherin in α-actinin-4 knockdown cells (yellow arrowheads). (L) BSA flux assays showing that tension-induced barrier enhancement is absent in α-actinin-4 and synaptopodin knockdown monolayers. (M) BSA flux assays showing decreased ability to withstand mechanical insult in α-actinin-4 and synaptopodin knockdown monolayers. (H, L, and M) Error bars are standard errors. n = 3. (N) Western blots showing E-cadherin, occludin, β-catenin, and p120-catenin levels unchanged in synaptopodin and α-actinin-4 knockdown cells. Bars: (A, D, G, I, and K) 10 µm.

Article Snippet: The coding sequence of human synaptopodin cDNAs (OriGene catalog no. RC228616) corresponding to aa 1–929, aa 1–600, aa 245–600, and aa 600–929 were subcloned into Sal1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with CBP and SBP tags at the N terminus for expression in MDCK cells. shRNAs for canine α-actinin-4 (5′-AGGTCCTGTTCCTCTGACTCGGTATCTAT-3′) and canine synaptopodin (5′-GAGGTGAGATCCAGCACACTTCTGATTGA-3′) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS and puromycin-selectable pRS vectors, respectively (OriGene).

Techniques: Quantitation Assay, Western Blot, Knockdown

Journal: The Journal of Cell Biology

Article Title: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

doi: 10.1083/jcb.201412003

Figure Lengend Snippet: Synaptopodin is required for mechanotransduction at the cell junction. (A) Deconvolved optical section at the apical junction showing tension-induced α-actinin-4 recruitment (yellow arrowheads) is reduced in synaptopodin knockdown monolayers (white arrowheads). (B) Quantitation of junctional synaptopodin and α-actinin-4 in A before (U) and after (P) cyclic pressure. Means are represented by horizontal lines. (C) Plotting junctional levels of synaptopodin and α-actinin-4 before (no P) and after (Low P) cyclic pressure shows similar slope in tension-induced response in parental and synaptopodin knockdown cells. (A–C) The images and quantitation shown are from a single representative experiment out of three experiments. (D) Wide-field immunofluorescence images showing cytoplasmic localization of vinculin in maturing synaptopodin knockdown cells before and after cyclic pressure. Junctional localization of vinculin in mature synaptopodin knockdown monolayers is also compromised. (E) Western blots showing that redistribution of vinculin and α-actinin-4 to the TX-100–insoluble fraction induced by intercellular tension is compromised in maturing synaptopodin knockdown cells. (F) Deconvolved z-composites showing spreading of synaptopodin knockdown cells on collagen-coated polyacrylamide gel. (G) Measurements of wound diameters showing wound-induced contractility is absent in synaptopodin and α-actinin-4 monolayers. Error bars are standard errors. n = 3. (H) Phase-contrast image of wound edge of MDCK cells after 30 min of wound expansion. (I) Dark-field images of wound showing expanded wound in WT but not in α-actinin-4 knockdown cells. Bars: (A, D, and F) 10 µm; (H) 50 µm; (I) 500 µm.

Article Snippet: The coding sequence of human synaptopodin cDNAs (OriGene catalog no. RC228616) corresponding to aa 1–929, aa 1–600, aa 245–600, and aa 600–929 were subcloned into Sal1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with CBP and SBP tags at the N terminus for expression in MDCK cells. shRNAs for canine α-actinin-4 (5′-AGGTCCTGTTCCTCTGACTCGGTATCTAT-3′) and canine synaptopodin (5′-GAGGTGAGATCCAGCACACTTCTGATTGA-3′) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS and puromycin-selectable pRS vectors, respectively (OriGene).

Techniques: Knockdown, Quantitation Assay, Immunofluorescence, Western Blot

Journal: The Journal of Cell Biology

Article Title: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

doi: 10.1083/jcb.201412003

Figure Lengend Snippet: Mechanical force induces Akt S473 phosphorylation that is compromised in synaptopodin knockdown monolayers. (A) Site-specific phospho-kinase arrays (see Materials and methods for details) showing induction of Akt S473 phosphorylation by cyclic pressure that is compromised in synaptopodin knockdown cells. (B) Quantitation of A showing fivefold increase of Akt S473 phosphorylation in WT MDCK but only threefold increase in synaptopodin knockdown cells. Error bars are standard errors. n = 2. (C) Wide-field immunofluorescence images showing tension-induction accumulation of phospho-Akt S473 at cell junction. Bar, 50 µm. (D and E) Line scans of C showing redistribution and colocalization of E-cadherin, α-actinin-4, and p-Akt S473 after cyclic pressure. (C–E) The images and quantitation shown are from a single representative experiment out of four experiments.

Article Snippet: The coding sequence of human synaptopodin cDNAs (OriGene catalog no. RC228616) corresponding to aa 1–929, aa 1–600, aa 245–600, and aa 600–929 were subcloned into Sal1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with CBP and SBP tags at the N terminus for expression in MDCK cells. shRNAs for canine α-actinin-4 (5′-AGGTCCTGTTCCTCTGACTCGGTATCTAT-3′) and canine synaptopodin (5′-GAGGTGAGATCCAGCACACTTCTGATTGA-3′) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS and puromycin-selectable pRS vectors, respectively (OriGene).

Techniques: Phospho-proteomics, Knockdown, Quantitation Assay, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

doi: 10.1083/jcb.201412003

Figure Lengend Snippet: Synaptopodin consists of three functional domains. (A) Western blot showing coimmunoprecipitation of synaptopodin internal aa 245–600 with endogenous synaptopodin, β-catenin, and α-actinin-4, whereas synaptopodin C-terminal aa 600–929 disrupted interactions of endogenous synaptopodin, β-catenin, and α-actinin-4. (B) Wide-field immunofluorescence images showing junctional localization of synaptopodin N-terminal aa 1–600 and C-terminal aa 600–929 in mature monolayers. However, only aa 1–600 redistributed from cell junction to cytoplasm when cells were grown on soft silicone substrates (white asterisks). (C) Wide-field immunofluorescence images showing synaptopodin FL aa 1–929 (blue arrowheads) and N-terminal aa 1–600 (white arrowheads) targeting to the cell junction in response to cyclic pressure. Expression of synaptopodin C-terminal aa 600–929, which colocalizes with α-actinin-4 (pink arrowheads), caused cells to round up when mechanical force was applied to the junction (yellow arrowheads). (D) Coomassie blue staining showing a major band copurifies with the synaptopodin C-terminal aa 600–929 fragment from MDCK cells. Mass spectroscopy identifies the band as myosin II. (E) The three domains of synaptopodin. Bars, 10 µm.

Article Snippet: The coding sequence of human synaptopodin cDNAs (OriGene catalog no. RC228616) corresponding to aa 1–929, aa 1–600, aa 245–600, and aa 600–929 were subcloned into Sal1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with CBP and SBP tags at the N terminus for expression in MDCK cells. shRNAs for canine α-actinin-4 (5′-AGGTCCTGTTCCTCTGACTCGGTATCTAT-3′) and canine synaptopodin (5′-GAGGTGAGATCCAGCACACTTCTGATTGA-3′) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS and puromycin-selectable pRS vectors, respectively (OriGene).

Techniques: Functional Assay, Western Blot, Immunofluorescence, Expressing, Staining, Mass Spectrometry

Journal: Scientific reports

Article Title: The potential roles of NAD(P)H:quinone oxidoreductase 1 in the development of diabetic nephropathy and actin polymerization.

doi: 10.1038/s41598-020-74493-z

Figure Lengend Snippet: Figure 3. NQO1 deficiency results in increased ACR and abnormal synaptopodin localization in response to STZ or LPS. (a) Urine albumin-to-creatinine ration (ACR) of WT mice and NKO mice at 0 (WT, n = 6; NKO, n = 6), 2 (WT, n = 3; NKO, n = 4), 4 (WT, n = 7; NKO; n = 7), 6 (WT, n = 5; NKO, n = 7), 8 weeks (WT, n = 5; NKO, n = 7) after STZ treatment was measured. *P < 0.05, **P < 0.01. (b) Urine ACR of WT mice (vehicle, n = 4; LPS, n = 7) and NKO mice (vehicle, n = 6; LPS, n = 7) is shown at 1 day post-LPS administration (15 mg/kg). *P < 0.05. (c) The numbers of WT-1 positive cell and the distribution of synaptopodin were estimated by immunostaining (× 60 magnification) using specific antibodies against WT1 (red) and synaptopodin (green). Scale bar is 20 μm. Fluorescence intensity of synaptopodin was analyzed using ImageJ software program (1.43u, https://imagej.nih. gov). *P < 0.05, **P < 0.01.

Article Snippet: Antibodies against synaptopodin were purchased from Novus Biologicals (for western blot, Centennial, CO, USA) or Progen (for immunostaining, Wayne, PA, USA).

Techniques: Immunostaining, Fluorescence, Software