Journal: Cell death & disease

Article Title: Sirtuin4 alleviates severe acute pancreatitis by regulating HIF-1α/HO-1 mediated ferroptosis.

doi: 10.1038/s41419-023-06216-x

Figure Lengend Snippet: Fig. 6 SIRT4 regulated ferroptosis in vivo and in vitro. A–B Representative IHC staining of 4-HNE in the pancreas of mice. Scale bar = 100 µm. C–D Western blot of different types of ferroptosis-related proteins (ACSL4, GPX4, SLC7A11) in pancreas of mice. E–F The protein expression level of ferroptosis-related proteins (ACSL4, GPX4, SLC7A11) in AR42J cells. A–F used GAPDH as the reference protein. *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.

Article Snippet: Primary antibodies were purchased from the following companies: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), heme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11) and Hypoxia-inducible factor-1α (HIF-1α) (Proteintech, Wuhan, China), NADPH-quinone oxidoreductase1 (NQO-1) (HuaBio, Hangzhou, China), glutathione peroxidase4 (GPX4), acylCoA synthetase long-chain family member4 (ACSL4) and Sirtuin4 (SIRT4) (abclonal, Wuhan, China), lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO) (Abcam, Shanghai, China), cluster of differentiation molecule 11b (CD11b) (Boster, Wuhan, China), 4-hydroxynoneral (4-HNE)(RD systems, Shanghai, China).

Techniques: In Vivo, In Vitro, Immunohistochemistry, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Sirtuin4 alleviates severe acute pancreatitis by regulating HIF-1α/HO-1 mediated ferroptosis.

doi: 10.1038/s41419-023-06216-x

Figure Lengend Snippet: Fig. 7 SIRT4 mitigated SAP by suppressing ferroptosis. A Fer-1 decreased the serum amylase level and lipase activity of SAP mice induced by L-Arg. B Representative HE staining images of pancreas. Scale bar = 100 µm. C The levels of oxidative stress (GSH, H2O2, SOD, MDA) in the pancreas of mice. D Representative IHC staining of CD11b, MPO, Ly6G and 4-HNE in the pancreas of mice in different groups. Scale bar = 100 µm. E The protein expression level of ferroptosis-related proteins (ACSL4, GPX4, SLC7A11) in the pancreas of mice in different groups. WT + SAP group, SIRT4_KO + SAP group, SIRT4_KO+Fer-1 + SAP group. E used GAPDH as the reference protein. *P < 0.05, **P < 0.01, ***P < 0.001. n = 6 per group.

Article Snippet: Primary antibodies were purchased from the following companies: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), heme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11) and Hypoxia-inducible factor-1α (HIF-1α) (Proteintech, Wuhan, China), NADPH-quinone oxidoreductase1 (NQO-1) (HuaBio, Hangzhou, China), glutathione peroxidase4 (GPX4), acylCoA synthetase long-chain family member4 (ACSL4) and Sirtuin4 (SIRT4) (abclonal, Wuhan, China), lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO) (Abcam, Shanghai, China), cluster of differentiation molecule 11b (CD11b) (Boster, Wuhan, China), 4-hydroxynoneral (4-HNE)(RD systems, Shanghai, China).

Techniques: Activity Assay, Staining, Immunohistochemistry, Expressing

Journal: Frontiers in Pharmacology

Article Title: Notoginsenoside R1 Regulates Ischemic Myocardial Lipid Metabolism by Activating the AKT/mTOR Signaling Pathway

doi: 10.3389/fphar.2022.905092

Figure Lengend Snippet: Validation of key differential protein expression in the lipid metabolism–associated network. (A) Heatmap of differential proteins in the lipid metabolism–associated network. (B) Expression of COX2, CPT2, ACADVL, and CD36 by Western blots. Values expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the MI group; # p < 0.05, ## p < 0.01, and #### p < 0.001 vs. the sham group ( n = 3 for sham, MI, NGR1-L, and NGR1-H groups).

Article Snippet: The primary antibodies against very long-chain specific acyl-CoA dehydrogenase (ACADVL) (Cat# PB1076) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat# BA2913) were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Biomarker Discovery, Expressing, Western Blot

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 1 PIK3R3 expression is regulated by nutritional status and is upregulated in HFD-induced fatty liver mice. (a) Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained liver sections from C57BL/6J mice fed a normal chow or high-fat diet (HFD) for 8 weeks (n = 6/group). (b) Serum ketone body (top panel) and hepatic TG (bottom panel) levels of mice fed a normal chow diet or HFD. (c) Quantitative PCR (top panel) analysis and western blot (bottom panel) analysis showing the expression level changes of Pik3r3 in mice livers during HFD feeding. (d) Quantitative PCR (left) analysis and western blot (right) analysis showing expression levels of hepatic Pik3r3 in C57BL/6J mice subjected to ad libitum feeding, 24 h fasting and 24 h fasted/24 h re-fed conditions (n = 2/group). Data are expressed as the means ± s.e.m. *Po0.05; **Po0.01.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

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

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 2 Overexpression of PIK3R3 regulated lipid metabolism in vitro and alleviated the fatty liver phenotype in vivo. (a) The indicated protein expression levels were detected by western blot after hepatic HepG2 and LO2 cells were transfected with the PIK3R3 overexpression vector (PIK3R3) or control vector (Vector). (b) Quantitative PCR analysis and chemical colorimetric diagnostic analysis showing the levels of genes involved in fatty acid oxidation in mice livers. (c) Mice were fed HFD for 8 weeks (n = 6/group) and injected with Ad-control or Ad-Pik3r3 as described in the Materials and methods. Mice were killed 5 days after the last injection. Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained sections of mouse livers. (d, e) Serum ketone body levels (d) and hepatic TG levels (e). Data are expressed as the means ± s.e.m. *Po0.05.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: Over Expression, In Vitro, In Vivo, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Diagnostic Assay, Injection, Staining

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 3 Down-regulation of PIK3R3 impairs lipid homeostasis in vitro and in vivo. (a) PIK3R3 was knocked down in hepatic HepG2 and LO2 cells by siRNA transfection; the indicated protein expression levels were detected by western blot. (b) Quantitative PCR analysis and chemical colorimetric diagnostic analysis showing the levels of genes involved in fatty acid oxidation in mice livers. (c) Mice were fed HFD for 8 weeks (n = 6/group) and injected with si-control or si-Pik3r3 as described in the Materials and methods. Mice were killed 5 days after the last injection. Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained sections of mouse livers. (d, e) Serum ketone body levels (d) and hepatic TG levels (e). Data are expressed as the means ± s.e.m. *Po0.05.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: In Vitro, In Vivo, Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Diagnostic Assay, Injection, Control, Staining

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 5 PPARα mediates the effects of PIK3R3 on cellular and hepatic lipid metabolism. (a) PIK3R3 was overexpressed in LO2 cells with or without the knockdown of PPARA; western blot analysis showing the protein levels of PIK3R3, PPARA, ACADM and CPT1A. (b) PIK3R3 was knocked down in HepG2 cells with or without the overexpression of PPARA; western blot analysis showing the protein levels of PIK3R3, PPARA, ACADM and CPT1A. (c) Mice were fed HFD for 8 weeks (n = 6/group) and injected with Ad-control, Ad-Pik3r3 or Ad-Pik3r3+si-Pparα as described in the Materials and methods. Mice were killed for further analysis 5 days after the last injection. Representative H&E, Oil Red O, Pparα, Acadm and Cpt1a stained sections of mouse livers. (d) Pik3r3 and Pparα expression levels were detected by western blot. (e) Chemical colorimetric diagnostic analysis showing the changes in hepatic TG. (f) Chemical colorimetric diagnostic analysis showing changes in serum ketone bodies. Data are expressed as the means ± s.e.m. *Po0.05.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: Knockdown, Western Blot, Over Expression, Injection, Control, Staining, Expressing, Diagnostic Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy

doi: 10.3892/etm.2016.4014

Figure Lengend Snippet: Atomic force microscopy imaging of IgG-SPA-CG on mica. Uniform globular structures of IgG-SPA-CG immune complexes were observed on mica (indicated by arrows). IgG-SPA-CG, colloidal gold-labeled staphylococcal protein A complexed with vascular endothelial growth factor receptor 2 monoclonal antibody.

Article Snippet: The following reagents were used: Rabbit anti-rat VEGFR-2 monoclonal antibody (BA0486; Wuhan Boster Bioengineering Co., Ltd.); M199 medium, fetal bovine serum, VEGF, double antibiotic solution (penicillin, streptomycin) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA); bradykinin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany); acetone (Guangzhou Weijia Technology Co., Ltd., Guangzhou, China); sorafenib (Guangzhou Baiji Pharmacy, Guangzhou, China); and colloidal gold-labeled staphylococcal protein A (SPA-CG; CG diameter 15 nm; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China).

Techniques: Microscopy, Imaging, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy

doi: 10.3892/etm.2016.4014

Figure Lengend Snippet: Atomic force microscopy imaging of the distribution of VEGFR-2 immune complexes on the glioma microvascular endothelial cell surface in the control group. Numerous globular structures were evenly distributed on the endothelial cell surface (indicated by arrows). VEGFR-2, vascular endothelial growth factor receptor 2; IgG-SPA-CG, colloidal gold-labeled staphylococcal protein A complexed with VEGFR-2 monoclonal antibody.

Article Snippet: The following reagents were used: Rabbit anti-rat VEGFR-2 monoclonal antibody (BA0486; Wuhan Boster Bioengineering Co., Ltd.); M199 medium, fetal bovine serum, VEGF, double antibiotic solution (penicillin, streptomycin) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA); bradykinin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany); acetone (Guangzhou Weijia Technology Co., Ltd., Guangzhou, China); sorafenib (Guangzhou Baiji Pharmacy, Guangzhou, China); and colloidal gold-labeled staphylococcal protein A (SPA-CG; CG diameter 15 nm; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China).

Techniques: Microscopy, Imaging, Control, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy

doi: 10.3892/etm.2016.4014

Figure Lengend Snippet: Atomic force microscopy imaging of the distribution of VEGFR-2 immune complexes on the glioma microvascular endothelial cell surface in the sorafenib group. Only a few globular structures were scattered on the endothelial cell surface (indicated by arrows). VEGFR-2, vascular endothelial growth factor receptor 2; IgG-SPA-CG, colloidal gold-labeled staphylococcal protein A complexed with VEGFR-2 monoclonal antibody.

Article Snippet: The following reagents were used: Rabbit anti-rat VEGFR-2 monoclonal antibody (BA0486; Wuhan Boster Bioengineering Co., Ltd.); M199 medium, fetal bovine serum, VEGF, double antibiotic solution (penicillin, streptomycin) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA); bradykinin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany); acetone (Guangzhou Weijia Technology Co., Ltd., Guangzhou, China); sorafenib (Guangzhou Baiji Pharmacy, Guangzhou, China); and colloidal gold-labeled staphylococcal protein A (SPA-CG; CG diameter 15 nm; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China).

Techniques: Microscopy, Imaging, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy

doi: 10.3892/etm.2016.4014

Figure Lengend Snippet: Atomic force microscopy imaging of the distribution of VEGFR-2 immune complexes on the glioma microvascular endothelial cell surface in the bradykinin group. Numerous globular structures were densely distributed on the endothelial cell surface (indicated by arrows). VEGFR-2, vascular endothelial growth factor receptor 2; IgG-SPA-CG, colloidal gold-labeled staphylococcal protein A complexed with VEGFR-2 monoclonal antibody.

Article Snippet: The following reagents were used: Rabbit anti-rat VEGFR-2 monoclonal antibody (BA0486; Wuhan Boster Bioengineering Co., Ltd.); M199 medium, fetal bovine serum, VEGF, double antibiotic solution (penicillin, streptomycin) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA); bradykinin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany); acetone (Guangzhou Weijia Technology Co., Ltd., Guangzhou, China); sorafenib (Guangzhou Baiji Pharmacy, Guangzhou, China); and colloidal gold-labeled staphylococcal protein A (SPA-CG; CG diameter 15 nm; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China).

Techniques: Microscopy, Imaging, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet

doi: 10.1016/j.jbc.2022.102648

Figure Lengend Snippet: The loss of pyruvate carboxylase elicits mitochondrial protein hyperacetylation. A , Western blot of acetylated proteins from 9-week-old fed and 24 h fasted Pcx f/f and Pcx L−/− mice. B , Western blot of acetylated proteins from 9-week-old fed, 24 h fasted, 12 week high fat diet–fed, and 1 week ketogenic diet–fed Pcx f/f and Pcx L−/− mice. C , quantitative TMT acetyl-proteomics of 24 h fasted Pcx f/f and Pcx L−/− livers showing mitochondrial proteins (n = 5). D , quantitative TMT acetyl-proteomics of 24 h fasted Pcx f/f and Pcx L−/− livers showing all proteins (n = 5). E , Western blots of mitochondrial proteins from 9-week-old Pcx f/f and Pcx L−/− mice (n = 4). Pyruvate Carboxylase (Pcx), Pyruvate Dehydrogenase 1a (Pdh1a), Citrate Synthase (Cs), Malate Dehydrogenase 2 (Mdh2), Mitochondrial Pyruvate Carrier 2 (Mpc2), Glutamine Oxaloacetate Transaminase 2 (Got2), Carnitine Palmitoyltransferase 2 (Cpt2), Acyl-CoA Dehydrogenase Medium Chain (Acadm), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha (Hadha), Acyl-CoA Thioesterase 2 (Acot2), ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 (Atp5a), ubiquinol cytochrome c reductase core protein 2 (Uqcrc2), mitochondrially encoded cytochrome c oxidase I (Mtco1), succinate dehydrogenase complex, subunit B (Sdhb), NADH:ubiquinone oxidoreductase subunit B8 (Ndufb8).

Article Snippet: Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) for 1 h and incubated overnight at 4 °C with primary antibodies at 1:1000 in 3% bovine serum albumin in TBST and all secondaries were diluted 1:5000 in 5% nonfat milk in TBST for 1 h. Antibodies used were as follows: pyruvate carboxylase (#16588-1-AP, Proteintech), HSC 70 (#sc-7298, Santa Cruz Biotechnology), pyruvate dehydrogenase (#2784 Cell Signaling), Citrate Synthase (#14309 Cell Signaling), Malate Dehydrogenase 2 (#11908 Cell Signaling), Mitochondrial Pyruvate Carrier 2 , Glutamine Oxaloacetate Transaminase 2 (HPA018139 Millipore Sigma), Carnitine Palmitoyltransferase 2 (PA5-12217 Thermo Fisher Scientific), Acyl-CoA Dehydrogenase Medium Chain (Genetex Irvine), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha (GTX101177 Genetex Irvine), Acyl-CoA Thioesterase 2 (15633-1-AP, Proteintech), Total Oxphos antibody cocktail (ab110411, Abcam), Acetylated-Lysine (#9441, Cell Signaling), mouse IgG-horseradish peroxidase (#7076, Cell Signaling), horseradish peroxidase–conjugated anti-rabbit (#NA934V Cytiva Life Sciences).

Techniques: Western Blot