Journal: Redox Biology

Article Title: Epigenetic regulation of TXNIP-mediated oxidative stress and NLRP3 inflammasome activation contributes to SAHH inhibition-aggravated diabetic nephropathy

doi: 10.1016/j.redox.2021.102033

Figure Lengend Snippet: SAHH inhibition increases high glucose-induced podocyte injury and aggravates STZ-induced diabetic nephropathy . (A) Podocytes were incubated with HG (20 mM) or ADA (20 μmol/L) for 24 h. F-actin was stained with rhodamine–phalloidin and examined by confocal laser scanning microscopy. Scale bar, 50 μm. (B) The protein expression of Desmin and Synaptopodin were assayed by Western blot in podocytes. Values are presented as mean ± SEM; n = 3, * P < 0.05 vs. control group; # P < 0.05 vs. HG group (determined by one-way ANOVA). (C) The activity of SAHH in kidney tissues was assayed by using a commercial fluorometric kit. (D – E) Urine albumin was measured using an ELISA kit, plasma and urine creatinine was measured using a colorimetric assay kit. (F) Immunofluorescence staining of SAHH and podocyte marker nephrin in kidney tissues. Scale bar, 20 μm. (G) Glomerular morphological changes were detected using Periodic Acid Schiff (PAS) staining. Scale bar, 20 μm. (H) Representative images and quantitation of podocyte ultrastructure changes by transmission electron microscopy (TEM) examination. Scale bar, 2 μm. (I) Representative immunofluorescence images and quantitation of WT1+ podocytes in glomeruli. Scale bar, 20 μm. Results are mean ± SEM; n = 6–10, * P < 0.05 vs. Ctrl group; # P < 0.05 vs. STZ group (determined by one-way ANOVA).

Article Snippet: Membranes were then incubated overnight at 4 °C with the primary antibodies to desmin (1:500) (Santa Cruz), synaptopodin (1:500) (Santa Cruz), NLRP3 (1:300) (Abcam), cleaved caspase-1 (1:500) (Cell signaling technology), cleaved gasdermin D (1:500) (Cell signaling technology), TXNIP (1:400) (Abcam), EGR1 (1:500) (Cell signaling technology), EZH2 (1:500) (Cell signaling technology), H3K27me3 (1:500) (Cell signaling technology), β-actin (1:2000) (Abcam), and histone H3 (1:200) (Cell signaling technology).

Techniques: Inhibition, Incubation, Staining, Confocal Laser Scanning Microscopy, Expressing, Western Blot, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Colorimetric Assay, Immunofluorescence, Marker, Quantitation Assay, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: Epigenetic regulation of TXNIP-mediated oxidative stress and NLRP3 inflammasome activation contributes to SAHH inhibition-aggravated diabetic nephropathy

doi: 10.1016/j.redox.2021.102033

Figure Lengend Snippet: SAHH inhibition increases high glucose-induced NLRP3 inflammasome activation in vitro and in vivo . (A) The protein expression of NLRP3 was assayed by Western blot in kidney tissues and podocytes. (B) Representative immunohistochemistry staining images and quantitation of NLRP3 expression in kidney tissues sections. Scale bar, 20 μm. (C) The mRNA levels of NLRP3 were measured by qRT-PCR in vivo . (D) IL-1β production and caspase-1 activity in supernatant of podocytes and kidney tissues was measured by ELISA and commercial kits. (E) The protein expression of NLRP3 was assayed by Western blot in podocytes incubated with ADA (20 μmol/L) in the presence or absence of NLRP3 siRNA for 48 h. (F) The protein expression of densmin and synaptopodin were assayed by Western blot in podocytes incubated with ADA (20 μmol/L) in the presence or absence of NLRP3 siRNA or caspase-1 inhibitor (Z-YVAD) for 48 h. (G) F-actin was stained with rhodamine–phalloidin and examined by confocal laser scanning microscopy. Scale bar, 50 μm. Values are presented as mean ± SEM; n = 3, for in vitro assays and n = 6–8 for in vivo assays, * P < 0.05 vs. control group; # P < 0.05 vs. ADA or HG (determined by one-way ANOVA or unpaired Student's t -test).

Article Snippet: Membranes were then incubated overnight at 4 °C with the primary antibodies to desmin (1:500) (Santa Cruz), synaptopodin (1:500) (Santa Cruz), NLRP3 (1:300) (Abcam), cleaved caspase-1 (1:500) (Cell signaling technology), cleaved gasdermin D (1:500) (Cell signaling technology), TXNIP (1:400) (Abcam), EGR1 (1:500) (Cell signaling technology), EZH2 (1:500) (Cell signaling technology), H3K27me3 (1:500) (Cell signaling technology), β-actin (1:2000) (Abcam), and histone H3 (1:200) (Cell signaling technology).

Techniques: Inhibition, Activation Assay, In Vitro, In Vivo, Expressing, Western Blot, Immunohistochemistry, Staining, Quantitation Assay, Quantitative RT-PCR, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, Confocal Laser Scanning Microscopy, Control

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: BMC nephrology

Article Title: The glomerular parietal epithelial cell's responses are influenced by SM22 alpha levels.

doi: 10.1186/1471-2369-15-174

Figure Lengend Snippet: Figure 2 Transition cells in SM22 null mice are higher with experimental glomerular disease compared to wildtypes. A) Representative images of transition cells at × 630 original magnification. Glomerular epithelial transition cells defined PAX2 (red, nuclear) and SYNPO (green, cytoplasmic) double positive cells. SM22α +/+ is A1-A9. SM22α −/−is A10-A18. Day 0 (A1-A3, A10-A12), day 7 (A4-A6, A13-A15), day 14 (A7-A9, A16-A18) were reacted anti PAX2 antibody (Ab) (A1, A4, A7, A10, A13, A16) and anti Synaptopodin (SYNPO) Ab (A2, A5, A8, A11, A14, A17). The images were merged (A3, A6, A9, A12, A15, A18). The glomerular transition cells are on Bowman’s capsule (arrow) and in tuft (arrow head). B) Number of glomerular epithelial transition cells along BBM / Bowman’s capsule (BC) length (mm) is increased in SM22α −/−mice, compared with SM22α +/+ mice. C) Number of glomerular epithelial transition cells in tuft area / tuft area (μm2) is increased in SM22α −/+ −mice, compared with SM22α +/+ mice.

Article Snippet: The following primary antibodies were used: rabbit anti-rat paired box gene 2 (PAX2, a PEC nuclear protein) polyclonal antibody diluted 1:500 (Zymed Laboratories, South San Francisco, Calif., USA) and mouse anti- Synaptopodin (Synpo, a podocyte protein) monoclonal antibody, diluted 1:10 (Fitzgerald).

Techniques:

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: 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

Journal: Journal of the American Heart Association

Article Title: Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co‐Occurrence of P63A and P380S BAG3 Variants

doi: 10.1161/jaha.123.029938

Figure Lengend Snippet: Figure 3. Assessment of BAG3-binding partners in the soluble protein fraction. A, Western blot for BAG3 and HSPB8 in the triton-soluble protein fraction with corresponding Revert total protein stain. B, Western blot for SYNPO2, HSP70, and CRYAB in the triton-soluble protein fraction with corresponding Revert total protein stain. C through G, Quantitative densitometry analysis of BAG3 (C), HSPB8 (D), SYNPO2 (E), HSP70 (F), and CRYAB (G) signals normalized to total protein signal. For all, n=4 NF, 5 DCM, 2 BAG363/380; data are presented as the mean±standard error and were analyzed by 1-way permutational ANOVA. CRYAB indicates crystallin alpha B; DCM, dilated cardiomyopathy; HSP70/B8, heat shock protein 70/β-8; NF, nonfailing; and SYNPO2, synaptopodin 2.

Article Snippet: The blot was incubated overnight at 4 °C with agitation in blocking solution with primary antibody: BAG3 (Proteintech, 10 599- 1- AP 1:4000), HSP70 (Proteintech, 10 995- 1- AP, 1:4000), HSPB8 (Proteintech, 15 287- 1- AP, 1:1000), Alpha B Crystallin (Proteintech, 15 808- 1- AP, 1:2500), SYNPO2 (synaptopodin- 2; Proteintech, 25 453- 1- AP 1:2500), Ubiquitin (Cytoskeleton Inc., AUB01, 1:500–1000), LC3 (CST, 2775S, 1:500–750), P62 (Proteintech, 18 420- 1- AP, 1:2500), Troponin I (IPOC, MA- 1040, 1:2000), pS23/24 Troponin I (CST, 1:1000), α- Actin (Sigma, A2172, 1:5000).

Techniques: Binding Assay, Western Blot, Staining

Journal: Journal of the American Heart Association

Article Title: Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co‐Occurrence of P63A and P380S BAG3 Variants

doi: 10.1161/jaha.123.029938

Figure Lengend Snippet: Figure 4. Assessment of BAG3-binding partners in the myofilament protein fraction. A, Western blot for BAG3 and HSPB8 in the myofilament protein fraction with corresponding Revert total protein stain. B, Western blot for SYNPO2, HSP70, and CRYAB in the myofilament protein fraction with corresponding Revert total protein stain. C through G, Quantitative densitometry analysis of BAG3 (C), HSPB8 (D), SYNPO2 (E), HSP70 (F), and CRYAB (G) signals normalized to total protein signal. For all, n=4 NF, 5 DCM, 2 BAG363/380; data are presented as the mean±standard error. CRYAB indicates crystallin alpha B; DCM, dilated cardiomyopathy; HSP70/B8, heat shock protein 70/β-8; NF, nonfailing; and SYNPO2, synaptopodin 2.

Article Snippet: The blot was incubated overnight at 4 °C with agitation in blocking solution with primary antibody: BAG3 (Proteintech, 10 599- 1- AP 1:4000), HSP70 (Proteintech, 10 995- 1- AP, 1:4000), HSPB8 (Proteintech, 15 287- 1- AP, 1:1000), Alpha B Crystallin (Proteintech, 15 808- 1- AP, 1:2500), SYNPO2 (synaptopodin- 2; Proteintech, 25 453- 1- AP 1:2500), Ubiquitin (Cytoskeleton Inc., AUB01, 1:500–1000), LC3 (CST, 2775S, 1:500–750), P62 (Proteintech, 18 420- 1- AP, 1:2500), Troponin I (IPOC, MA- 1040, 1:2000), pS23/24 Troponin I (CST, 1:1000), α- Actin (Sigma, A2172, 1:5000).

Techniques: Binding Assay, Western Blot, Staining

Journal: Cells

Article Title: Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in Cardiomyocytes

doi: 10.3390/cells13161373

Figure Lengend Snippet: SYNPO2L, α-actinin, and actin filaments accumulate at the Z-discs of the sarcomere in mouse cardiomyocytes. Double immunofluorescence images of α-actinin and actin filaments ( upper panels), SYNPO2L and actin filaments ( middle panels) or SYNPO2L and α-actinin ( bottom panels) in mouse cardiomyocytes. Areas indicated by rectangles are enlarged in the underneath image. Bar: 30 μm.

Article Snippet: Rabbit anti-SYNPO2L antibody (cat# 21480-1-AP) was purchased from Proteintech Group Inc. (Rosemont, IL, USA).

Techniques: Immunofluorescence

Journal: Cell death & disease

Article Title: Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.

doi: 10.1038/s41419-020-2481-5

Figure Lengend Snippet: Fig. 1 Metabolomics analysis revealed higher lactate production in differentiated podocytes. a Immunofluorescence staining for nephrin (red), phalloidine for F-actin (green) and DAPI for nuclear (blue) in undifferentiated podocytes (UDPs) or differentiated podocytes (DPs) as indicated (n = 5). Scale bar=5 μm. b Representative western blotting results of nephrin, podocin and synaptopodin confirm the differentiation of podocytes (n = 3). c Representative map of 1H-NMR spectra in the extracellular medium incubated with the presence of different podocytes (n = 6). UDM: undifferentiated podocyte medium; DM: differentiated podocyte medium. The map shows the significance of metabolites variations between these two classes. Peaks in the positive direction indicate metabolites that are more abundant in the UDM groups. Consequently, metabolites that are more abundant in DM are presented as peaks in the negative direction.

Article Snippet: Primary antibodies were detected against Nephrin (ab80298, Abcam), Podocin (P0372, Sigma), Synaptopodin (NBP239100, Novus), GLUT1 (ab115730, Abcam), GLUT4 (NBP1-49533, Novus), HK2 (NBP2-02272, Novus), F6PK (ab154804, Abcam), PKM1 (7067 S, CST), p-PKM2 (3827 S, CST), PKM2 (4053 S, CST), PKLR (ab137787, Abcam), LDHA (3558 S, CST), PGC-1α (ab54481, Abcam), TFAM (ab131607, Abcam), OPA-1 (ab42364, Official journal of the Cell Death Differentiation Association Abcam), DRP-1 (ab184247, abcam), VDAC (4866 s, cst), PCNA (ab18197, abcam), p-S6 (4858 s, cst), S6 (2217 s, cst), and Tubulin (T9026, Sigma) at a dilution of 1:1000.

Techniques: Staining, Western Blot, Incubation

Journal: Cell death & disease

Article Title: Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.

doi: 10.1038/s41419-020-2481-5

Figure Lengend Snippet: Fig. 8 mTOR signaling pathway regulated PKM2 expression and podocyte differentiation. a IHC detection of p-S6 in renal cortex during postnatal development. Scale bar=20 μm. b Representative western immunoblot analysis of p-S6 in renal cortex. c Representative western immunoblot analysis of p-S6 in cultured podocyte during differentiation. d Western immunoblot analysis of p-S6 and PKM2 expression in Tsc1fl/fland Tsc1−/−-podocytes. e Rapamycin caused a significant reduction in the protein levels of p-S6 and PKM2 in cultured podocyte. f Western immunoblot analysis of nephrin and synaptopodin with 10 nM rapamycin treatment for 14 days.

Article Snippet: Primary antibodies were detected against Nephrin (ab80298, Abcam), Podocin (P0372, Sigma), Synaptopodin (NBP239100, Novus), GLUT1 (ab115730, Abcam), GLUT4 (NBP1-49533, Novus), HK2 (NBP2-02272, Novus), F6PK (ab154804, Abcam), PKM1 (7067 S, CST), p-PKM2 (3827 S, CST), PKM2 (4053 S, CST), PKLR (ab137787, Abcam), LDHA (3558 S, CST), PGC-1α (ab54481, Abcam), TFAM (ab131607, Abcam), OPA-1 (ab42364, Official journal of the Cell Death Differentiation Association Abcam), DRP-1 (ab184247, abcam), VDAC (4866 s, cst), PCNA (ab18197, abcam), p-S6 (4858 s, cst), S6 (2217 s, cst), and Tubulin (T9026, Sigma) at a dilution of 1:1000.

Techniques: Expressing, Western Blot, Cell Culture