Journal: The Journal of Biological Chemistry

Article Title: Disruption of sirtuin 7 in zebrafish facilitates hypoxia tolerance

doi: 10.1016/j.jbc.2023.105074

Figure Lengend Snippet: Zebrafish sirt7 is suppressed by hypoxia. A , quantitative real-time PCR analysis (qPCR) of sirt7 , as well as well-defined hypoxia-responsive genes, including cited2 , pai1 , phd3 , vegfaa , and epoa , in zebrafish larvae (3 dpf) under normoxia (Nor) and hypoxia (Hyp). B , qPCR analysis of sirt7 , as well as well-defined hypoxia-responsive genes, including phd3 , cited2 , ldha , and vegfaa , in ZFL cells under normoxia (Nor) and hypoxia (Hyp). C , qPCR analysis of sirt7 in ZFL cells transfected with empty vector control (Flag empty) or Flag- hif-1αa , or Flag- hif-1αb , or Flag- hif-2αa , or Flag- hif-2αb under normoxia. D , qPCR analysis of phd3 in ZFL cells transfected with empty vector control (Flag empty) or Flag- hif-1αa , or Flag- hif-1αb , or Flag- hif-2αa , or Flag- hif-2αb under normoxia. E , qPCR analysis of cited2 in ZFL cells transfected with empty vector control (Flag empty) or Flag- hif-1αa , or Flag- hif-1αb , or Flag- hif-2αa , or Flag- hif-2αb under normoxia. F , qPCR analysis of ldha in ZFL cells transfected with empty vector control (Flag empty) or Flag- hif-1αa , or Flag- hif-1αb , or Flag- hif-2αa , or Flag- hif-2αb under normoxia. G , qPCR analysis of vegfaa in ZFL cells transfected with empty vector control (Flag empty) or Flag- hif-1αa , or Flag- hif-1αb , or Flag- hif-2αa , or Flag- hif-2αb under normoxia. p Values were calculated by unpaired Student's t test ( A and B ) or two-way ANOVA analysis ( C – G ); ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001; data based on one representative experiment performed in three biological replicates from at least three independent experiments (mean ± SD).

Article Snippet: Zebrafish liver (ZFL) cells (originally obtained from the American Type Culture Collection) were cultured in Ham’s F-12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Biological Industries).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Disruption of sirtuin 7 in zebrafish facilitates hypoxia tolerance

doi: 10.1016/j.jbc.2023.105074

Figure Lengend Snippet: Zebrafish sirt7 suppresses hypoxia-responsive gene expression. A , Western blot analysis of indicated protein levels in EPC cells transfected empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 (Myc- sirt7 ). B , luciferase activity of hypoxia-responsive element (HRE)-luciferase reporter in EPC cells transfected with empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 (Myc- sirt7 ) under normoxia (Nor) or hypoxia (Hyp). C , Western blot analysis of indicated protein levels in ZFL cells transfected empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 (Myc- sirt7 ). D , quantitative real-time PCR (qPCR) analysis of phd3 in ZFL cells transfected with empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 (Myc- sirt7 ) under normoxia (Nor) or hypoxia (Hyp). E , qPCR analysis of vegfaa in ZFL cells transfected with empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 (Myc- sirt7 ) under normoxia (Nor) or hypoxia (Hyp). F , qPCR analysis of ldha in ZFL cells transfected with empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 (Myc- sirt7 ) under normoxia (Nor) or hypoxia (Hyp). G , qPCR analysis of cited2 in ZFL cells transfected with empty vector control (Myc empty) or Myc-tagged zebrafish sirt7 under normoxia (Nor) or hypoxia (Hyp). p Values were calculated by two-way ANOVA analysis ( B , D , E , F , and G ); ∗∗∗∗ p < 0.0001; data based on one representative experiment performed in three biological replicates from at least three independent experiments (mean ± SD).

Article Snippet: Zebrafish liver (ZFL) cells (originally obtained from the American Type Culture Collection) were cultured in Ham’s F-12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Biological Industries).

Techniques: Gene Expression, Western Blot, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Disruption of sirtuin 7 in zebrafish facilitates hypoxia tolerance

doi: 10.1016/j.jbc.2023.105074

Figure Lengend Snippet: Zebrafish sirt7 attenuates hypoxia signaling independent of its deacetylase activity. A , Western blot analysis of indicated protein levels in EPC cells transfected with empty vector, wildtype sirt7 , or the enzyme-deficient mutants sirt 7 -S115A and sirt 7 -H191Y. B , luciferase activity of HRE-luciferase reporter in EPC cells transfected with empty vector, wildtype sirt7 , or the enzyme-deficient mutants sirt 7 -S115A and sirt 7 -H191Y under normoxia (Nor) or hypoxia (Hyp). C , Western blot analysis of indicated protein levels in ZFL cells transfected with empty vector, wildtype sirt7 , or the enzyme-deficient mutants sirt 7 -S115A and sirt 7 -H191Y. D , quantitative real-time PCR (qPCR) analysis of phd3 in ZFL cells transfected with empty vector, wildtype sirt7 , or the enzyme-deficient mutants sirt 7 -S115A and sirt 7 -H191Y under normoxia (Nor) or hypoxia (Hyp). E , qPCR analysis of cited2 in ZFL cells transfected with empty vector, wildtype sirt7 , or the enzyme-deficient mutants sirt 7 -S115A and sirt 7 -H191Y under normoxia (Nor) or hypoxia (Hyp). F , qPCR analysis of ldha in ZFL cells transfected with empty vector, wildtype sirt7 , or the enzyme-deficient mutants sirt 7 -S115A and sirt 7 -H191Y under normoxia (Nor) or hypoxia (Hyp). p Values were calculated by two-way ANOVA analysis ( B , D , E , and F ); ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001; data based on one representative experiment performed in three biological replicates from at least three independent experiments (mean ± SD).

Article Snippet: Zebrafish liver (ZFL) cells (originally obtained from the American Type Culture Collection) were cultured in Ham’s F-12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Biological Industries).

Techniques: Histone Deacetylase Assay, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase, Real-time Polymerase Chain Reaction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Pancreastatin inhibitor activates AMPK pathway via GRP78 and ameliorates dexamethasone induced fatty liver disease in C57BL/6 mice.

doi: 10.1016/j.biopha.2019.108959

Figure Lengend Snippet: Fig. 6. PSTi8 increases insulin sensitivity and regulates glucose metabolic genes. Protein expression in liver tissues were analysed by western blot after homogenising liver tissues of sacrificed mice. (a) pSer612 IRS-1, (b) IR-β, (c) pThr308 AKT, (d) pS256 FoxO1, (e) pS9 GSK3β. Similarly liver tissues were isolated after sacrifice of mice and subjected to mRNA extraction to determined expression of (f) PEPCK, G6Pases, GP and Ppar-α genes by using qRT-PCR. Protein expression of gluconeogenic genes (g) G6Pase, (h) FBPase, (i) PEPCK were analysed in liver tissue. All the bar graphs of western blot show densitometry of protein expression. The data were analysed, n = 6 mice per group. Data are represented as mean ± SEM. α, Control vs DEX; β DEX vs DEX + PSTi8; α, β indicates comparison between groups. Significance *p < 0.05; **p < 0.01; ***p < 0.001. DEX, dexamethasone; Ins, insulin; NS, non-significant.

Article Snippet: After immunobloting, blot was incubate with 5% (w/v) non-fat milk powder for 1 h. Blots was washed with TBST and subsequently treated overnight at 4 0C with primary antibodies such as anti phospho-AMPKα (#2535), AMPKα (#2532), phospho-Srebp-1c (#9874), phospho-Acc (#3661), Acc (#3662), phospho-NFkβp65 (#3031), TNFα (#3707), phospho-IRS-1 (#2386), IR-β (#3025), phospho-AKT (#9275), Pan AKT (#4691), phospho-FoxO1 (#9461), FoxO1 (#2880), phospho-GSK3β (#9336), GSK3β (#9315) and β-Actin (#3700) from Cell signalling Technology, Denvers, USA, G6Pase (sc398155), FBPase (sc-374396) and PEPCK (sc-271029) (Santa Cruz Biotechnology, Dallas, USA), UCP3 (E-AB-11774) (Elabscience, Houston, USA) and Gapdh (MAB374) (Merkmillipore, Burlington, USA) (1:1000).

Techniques: Expressing, Western Blot, Isolation, Extraction, Quantitative RT-PCR, Control, Comparison