Journal: Theranostics

Article Title: Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes

doi: 10.7150/thno.45003

Figure Lengend Snippet: Podocyte-specific deletion of Sirt6 aggravates Ang II-induced cholesterol accumulation in podocytes in vivo . (A) Representative microscopy images and quantification of WT1 and Adrp double staining of kidney sections for each group (original magnification×600). * P < 0.05, n=30. Scale bars: 20 µm. (B) Representative microscopy images and quantification of filipin and WT1 double staining of kidney sections in each group (original magnification ×600). * P < 0.05, n=30. Scale bars: 20 µm. (C) Representative Western blots and quantification of ABCA1, HMGCR and ABCG1 protein levels in each group. * P < 0.05, n=3. ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1; Adrp: adipocyte differentiation-related protein; HMGCR: 3-hydroxy-3-methylglutaryl coenzyme A reductase; LDLR: low-density lipoprotein receptor; Control: Sirt6 flox/flox /Nphs2.Cre - group; Sirt6.Podo-cKO: Sirt6 podocyte conditional knockout: Sirt6 flox/flox /Nphs2.Cre + group; WT1: Wilms' tumor-1.

Article Snippet: The frozen kidney sections were blocked and incubated with a mixture of guinea pig anti-adipocyte differentiation-related protein (Adrp) antibody (1:100, Progen Biotechnik, Germany) and mouse anti-WT1 antibody (1:100, Novus, USA) or a mixture of rabbit anti-Sirt6 antibody (1:100, Thermo Fisher Scientific, USA) and mouse anti-WT1 antibody (1:100, Novus, USA)/mouse anti-Synaptopodin antibody (1:100, Progen Biotechnik, Germany) overnight at 4 °C, followed by incubation with fluorescent secondary antibodies (1:200, Thermo Fisher Scientific, USA) at 37 °C for 90 min in the dark.

Techniques: In Vivo, Microscopy, Double Staining, Western Blot, Binding Assay, Knock-Out, Wilms Tumor Assay

Journal: Theranostics

Article Title: Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes

doi: 10.7150/thno.45003

Figure Lengend Snippet: Podocyte-specific deletion of Sirt6 affects the protective effect of cholesterol-lowering agents on Ang II-induced injury in podocytes. (A) Representative microscopy images and quantification of WT1 and Adrp double staining of kidney sections for each group (original magnification×600). * P < 0.05, n=30. Scale bars: 20 µm. (B) Representative microscopy images and quantification of filipin and WT1 double staining of kidney sections in each group (original magnification×600). * P < 0.05, n=30. Scale bars: 20 µm. (C) Quantitative analysis of body weight in different groups. * P < 0.05, n=6. (D) Quantitative analysis of ACR (albumin-to-creatinine ratio) in different groups. * P < 0.05, n=6. (E) Representative microscopy images and quantification of PAS of kidney sections for each group (original magnification ×400), Scale bars: 20 µm; Representative transmission electron microscopic images of the ultrastructure of capillary loops (original magnification×10,000) and quantitation of foot process effacement in each group, Scale bars: 2 µm. (F) Quantification of apoptotic podocytes of WT1 and TUNEL double staining of kidney sections for each group (original magnification×600). * P < 0.05, n=6. Control: Sirt6 flox/flox /Nphs2.Cre - group; cKO: Sirt6 podocyte conditional knockout: Sirt6 flox/flox /Nphs2.Cre + group; Ang II: Ang II-infused group; Ang II+SV: Ang II-infused and simvastatin administration group; Ang II+CD: Ang II-infused and CD administration group; CD: cyclodextrin; LDs: lipid droplets; TEM: transmission electron microscopy; WT1: Wilms' tumor-1.

Article Snippet: The frozen kidney sections were blocked and incubated with a mixture of guinea pig anti-adipocyte differentiation-related protein (Adrp) antibody (1:100, Progen Biotechnik, Germany) and mouse anti-WT1 antibody (1:100, Novus, USA) or a mixture of rabbit anti-Sirt6 antibody (1:100, Thermo Fisher Scientific, USA) and mouse anti-WT1 antibody (1:100, Novus, USA)/mouse anti-Synaptopodin antibody (1:100, Progen Biotechnik, Germany) overnight at 4 °C, followed by incubation with fluorescent secondary antibodies (1:200, Thermo Fisher Scientific, USA) at 37 °C for 90 min in the dark.

Techniques: Microscopy, Double Staining, Transmission Assay, Quantitation Assay, TUNEL Assay, Knock-Out, Electron Microscopy, Wilms Tumor Assay

Journal: Theranostics

Article Title: Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes

doi: 10.7150/thno.45003

Figure Lengend Snippet: Deletion of Sirt6 affects the protective effect of CD on Ang II-induced podocyte injury in vitro . Podocytes were transfected with scrambled siRNA, or Sirt6 siRNA before pretreatment with 5 mM CD for 1 h and then stimulated with 10 -7 M Ang II for 24 h. (A) Representative microscopy images and quantification of Adrp staining in each group (original magnification×600). * P < 0.05, n=20. Scale bars: 10 µm. (B) Representative microscopy images and quantification of filipin staining in each group (original magnification×600). * P < 0.05, n=20. Scale bars: 10 µm. (C) Quantitative analysis of cholesterol content in each group. * P < 0.05, n=9. (D) Quantitative analysis of cholesterol efflux rate in each group. * P < 0.05, n=6. (E) Flow cytometry analysis of the apoptotic rate of podocytes in each group. * P < 0.05, n=4. (F) Representative Western blots and quantification of ABCA1, HMGCR and ABCG1 protein levels in each group. * P < 0.05, n=3. ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1; Adrp: adipocyte differentiation-related protein; CD: cyclodextrin; HMGCR: 3-hydroxy-3-methylglutaryl coenzyme A reductase; LDLR: low-density lipoprotein receptor.

Article Snippet: The frozen kidney sections were blocked and incubated with a mixture of guinea pig anti-adipocyte differentiation-related protein (Adrp) antibody (1:100, Progen Biotechnik, Germany) and mouse anti-WT1 antibody (1:100, Novus, USA) or a mixture of rabbit anti-Sirt6 antibody (1:100, Thermo Fisher Scientific, USA) and mouse anti-WT1 antibody (1:100, Novus, USA)/mouse anti-Synaptopodin antibody (1:100, Progen Biotechnik, Germany) overnight at 4 °C, followed by incubation with fluorescent secondary antibodies (1:200, Thermo Fisher Scientific, USA) at 37 °C for 90 min in the dark.

Techniques: In Vitro, Transfection, Microscopy, Staining, Flow Cytometry, Western Blot, Binding Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effects of Dietary Supplementation of Lauric Acid on Lactation Function, Mammary Gland Development, and Serum Lipid Metabolites in Lactating Mice

doi: 10.3390/ani10030529

Figure Lengend Snippet: The effects of dietary supplementation of 1% LA on the relative gene expressions in the mammary glands of lactating mice. ( A ) The relative mRNA expression levels of FASN, PPARγ, FATP4, and ACADM in mammary glands (n = 4). ( B ) Western blot analysis of ADRP, β-casein, PRLR, and Elf5 in the mammary glands of lactating mice. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of ADRP, β-casein, PRLR, Elf5; the intensities of the bands are expressed as arbitrary units (n = 4). CD is control group. ** p < 0.01 versus control group.

Article Snippet: Polyclonal antibodies against β-casein, adipocyte differentiation-related protein (ADRP), E74-like factor 5 (Elf5), phospho-Akt Ser473 (p-Akt Ser473 ), Akt, and PRL receptor (PRLR) were bought from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effects of Dietary Supplementation of Lauric Acid on Lactation Function, Mammary Gland Development, and Serum Lipid Metabolites in Lactating Mice

doi: 10.3390/ani10030529

Figure Lengend Snippet: The effects of 100 μM LA on the content of TG and relative gene expressions in HC11 cells by DIP-induction. ( A ) The content of TG in HC11 cells and in the cell supernatant. ( B ) The relative mRNA expression level of FASN, PPARγ, FATP4, and ACADM in HC11 cells by DIP-induction (n = 4). ( C ) Western blot analysis of ADRP, β-casein, and Elf5 in HC11 cells by DIP- induction. β-actin was used as the loading control. ( D ) Mean ± SEM of immunoblotting bands of ADRP, β-casein Elf5, the intensities of the bands are shown as arbitrary units (n = 4). CD is control group. * p < 0.05 versus control group.

Article Snippet: Polyclonal antibodies against β-casein, adipocyte differentiation-related protein (ADRP), E74-like factor 5 (Elf5), phospho-Akt Ser473 (p-Akt Ser473 ), Akt, and PRL receptor (PRLR) were bought from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Lipofibroblast Phenotype in Pulmonary Interstitial Glycogenosis

doi: 10.1164/rccm.201509-1809le

Figure Lengend Snippet: Figure 1. Lipofibroblast phenotype in pulmonary interstitial glycogenosis. Characteristic spindle cells expand the alveolar interstitium in the biopsy from case 4, which also showed poor lung growth and hypertensive change of the pulmonary arteries (A). Transmission electron microscopy of interstitial cells in the alveolar wall from case 3 (B, original magnification, 38,500) and case 2 (C, original magnification 318,200), showing some of these cells contain lipid bodies (arrows), as well as dispersed cytoplasmic glycogen. Oil-red-O staining (D) demonstrates lipid droplets of variable size in scattered alveolar interstitial cells; cytokeratin (Dako M3515) immunostaining denotes epithelial cells. (E and F) Some interstitial cells in pulmonary interstitial glycogenosis express adipocyte differentiation–related protein (Fitzgerald 10R-A117ax; E developed with 3,39-diaminobenzidine; F developed with fluorescein isothiocyanate-green), which colocalizes with vimentin (red; Dako M7020; nuclei in blue, 49,6-diamidino-2-phenylindole counterstain). No significant oil-red-O positive or adipocyte differentiation-related protein immunopositive interstitial cells were seen within lung biopsies from age-matched patients without histologic findings of pulmonary interstitial glycogenosis (data not shown).

Article Snippet: Oil-red-O staining (D) demonstrates lipid droplets of variable size in scattered alveolar interstitial cells; cytokeratin (Dako M3515) immunostaining denotes epithelial cells. (E and F) Some interstitial cells in pulmonary interstitial glycogenosis express adipocyte differentiation–related protein (Fitzgerald 10R-A117ax; E developed with 3,39-diaminobenzidine; F developed with fluorescein isothiocyanate-green), which colocalizes with vimentin (red; Dako M7020; nuclei in blue, 49,6-diamidino-2-phenylindole counterstain).

Techniques: Transmission Assay, Electron Microscopy, Staining, Immunostaining

Journal: Journal of Lipid Research

Article Title: Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells [S]

doi: 10.1194/jlr.M046359

Figure Lengend Snippet: Atg2A localizes to ADRP-positive LDs independent of treatments that modulate autophagy. GFP-Atg2A/U2OS cells were incubated with or without OA for 24 h, followed by treatments with NF medium for 3 h. Subsequently, the cells were immunostained with anti-ADRP/IgG-conjugated Alexa 546 to detect endogenous ADRP and with TO-PRO-3 to mark cell nuclei, followed by confocal LSM (n = 3). A: Representative images from NF-treated cells. B: Representative images from OA + NF-treated cells. The cell boundaries are marked by a dotted line in the upper panels, and the typical ring-like ADRP-positive LD appearance is highlighted by further magnification in the lower panels. Scale bar, 20 µm. Supporting material is available (supplementary Figs. II and IIIA).

Article Snippet: Antibodies and fluorescent dyes The following primary antibodies were used in this study: anti-tubulin (B-5-1-2) (Cat. No. T5168, Sigma-Aldrich), anti- myc (9E10) (Cat. No. sc-40 or sc-789, Santa Cruz Biotechnology), anti-GFP antibody (Cat. No. 11814460001, Roche), anti-adipocyte differentiation-related protein (ADRP) (Cat. No. 610102, ProGen), anti-LC3 (Cat. No. 0231-100/LC3-5F10, Nano Tools), and anti-GAPDH (Cat. No. ACR001P, Acris).

Techniques: Incubation

Journal: Journal of Lipid Research

Article Title: Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells [S]

doi: 10.1194/jlr.M046359

Figure Lengend Snippet: OA treatment increased Atg2A targeting to ADRP-positive LDs. G361 cells transiently expressing GFP-Atg2A were incubated with or without OA for 24 h, followed by treatments with NF medium for 3 h. Cells were immunostained with anti-ADRP/IgG Alexa 546 antibodies analyzed by confocal LSM (n = 3). A: Image projections of confocal sections were quantified with regard to fluorescence areas by using Image Pro Plus, providing the percentage of the total inclusion area (ADRP or GFP-Atg2A vesicles) in relation to the total cell area. NF, n = 21 cells; OA + NF, n = 18 cells (from 3 independent experiments). B: Using the ImageJ colocalization threshold tool, the PCC for individual cells (n = 34 cells per treatment) was measured. The MOC for GFP-Atg2A overlapping with ADRP (C) and ADRP overlapping with GFP-Atg2A (D) was also measured using the ImageJ colocalization threshold. Mean ± SEM. P values: n.s. (not significant) P ≥ 0.05, *** P ≤ 0.001. Supporting material is available (supplementary Fig. IIIB).

Article Snippet: Antibodies and fluorescent dyes The following primary antibodies were used in this study: anti-tubulin (B-5-1-2) (Cat. No. T5168, Sigma-Aldrich), anti- myc (9E10) (Cat. No. sc-40 or sc-789, Santa Cruz Biotechnology), anti-GFP antibody (Cat. No. 11814460001, Roche), anti-adipocyte differentiation-related protein (ADRP) (Cat. No. 610102, ProGen), anti-LC3 (Cat. No. 0231-100/LC3-5F10, Nano Tools), and anti-GAPDH (Cat. No. ACR001P, Acris).

Techniques: Expressing, Incubation, Fluorescence

Journal: Journal of Lipid Research

Article Title: Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells [S]

doi: 10.1194/jlr.M046359

Figure Lengend Snippet: Density gradient centrifugation reveals the presence of ADRP, GFP-Atg2A, and myc-Atg14L in the floating LD fraction. Stable GFP-Atg2A/U2OS cells were transiently transfected with myc-Atg14L and incubated with OA for 24 h. Cell lysates were fractionated upon sucrose density centrifugation (n = 2). The floating LD fraction (boxed with a dotted red line) and intermediate and first cytoplasmic fractions were analyzed by Western blotting using anti-ADRP, anti-GFP, anti-myc, and anti-GAPDH antibodies.

Article Snippet: Antibodies and fluorescent dyes The following primary antibodies were used in this study: anti-tubulin (B-5-1-2) (Cat. No. T5168, Sigma-Aldrich), anti- myc (9E10) (Cat. No. sc-40 or sc-789, Santa Cruz Biotechnology), anti-GFP antibody (Cat. No. 11814460001, Roche), anti-adipocyte differentiation-related protein (ADRP) (Cat. No. 610102, ProGen), anti-LC3 (Cat. No. 0231-100/LC3-5F10, Nano Tools), and anti-GAPDH (Cat. No. ACR001P, Acris).

Techniques: Gradient Centrifugation, Transfection, Incubation, Centrifugation, Western Blot