pgl-3 promoter vector  (Promega)

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a Analysis of levels of the HIPK2 mRNA by Q-PCR; GAPDH was used as an internal control. The data are presented as means ± SEM ( n = 3), # p < 0.05 compared with the sham group. b , c Western blot analysis of levels of the HIPK2 protein ( b ) and the results of the corresponding semi-quantitative analysis of levels of the HIPK2 protein based on the optical density measured using ImageJ software ( c ); the data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the sham group. d , e Images of IHC staining for the HIPK2 protein ( d ) and the results of the corresponding semi-quantitative analysis of levels of the HIPK2 protein based on the optical density measured using ImageJ software ( e ). The data are presented as means ± SEM ( n = 3), # p < 0.05 compared with the sham group. The liver and serum samples were obtained from mice 16 h after the CLP surgery

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Western Blot, Software, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a HIPK2 was overexpressed following the injection of Ad-HIPK2 and HIPK2 was knocked down following the injection of Ad-shHIPK2, and hepatic HIPK2 expression was analysed by Q-PCR; the data are presented as means ± SEM ( n = 3), # p < 0.05 compared with the Ad-vector group. b The expression of HIPK2 in several different tissues was analysed by Q-PCR; the data are presented as means ± SEM ( n = 3), # p < 0.05 compared with the Ad-vector group. c Effect of HIPK2 on the survival of mice with CLP-induced sepsis; the data are presented as means ± SEM ( n = 30 mice per group), # p < 0.05 compared with the sham plus Ad-vector group, + p < 0.05 compared with the CLP plus Ad-vector group. In this section, the CLP was performed on the fifth day after the adenovirus injection, and the liver and serum were obtained 16 h after CLP-induced sepsis. d , e , f Effect of HIPK2 on serum AST, ALT, and ALP concentrations, as determined by ELISAs; the data are presented as means ± SEM ( n = 30 mice per group), # p < 0.05 compared with the sham plus Ad-vector group, + p < 0.05 compared with the CLP plus Ad-vector group. g , h , i Effect of HIPK2 on liver AST, ALT, and ALP levels, as analysed by ELISAs; the data are presented as means ± SEM ( n = 30 mice per group), # p < 0.05 compared with the sham plus Ad-vector group, + p < 0.05 compared with the CLP plus Ad-vector group

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Injection, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a , b Effects of HIPK2 on levels of the LC3-II and p62 proteins in the liver; the data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the control group and + p < 0.05 compared with the CLP plus Ad-vector group. c Effects of Ad-HIPK2 on hepatic LC3-II expression in of the group treated with bafilomycin A (0.2 μM); the data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the Ad-vector (control) group at the indicated time point. d Effect of chloroquine (CQ, 50 mg/kg per day) on levels of the LC3-II protein in the liver; the data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the sham plus Ad-vector group in the absence of CQ, + p < 0.05 compared with the CLP plus Ad-vector group in the absence of CQ, and @ p < 0.05 compared with the CLP plus Ad-HIPK2 group in the absence of CQ. e , f , g Effect of chloroquine (CQ) on serum concentrations of AST, ALT, and ALP in the HIPK2-overexpressing liver tissues, as determined by ELISAs; the data are presented as means ± SEM ( n = 30 mice per group), # p < 0.05 compared with the sham plus Ad-vector group in the absence of CQ, + p < 0.05 compared with the CLP plus Ad-vector group in the absence of CQ, and @ p < 0.05 compared with the CLP plus Ad-HIPK2 group in the absence of CQ. h Effect of chloroquine (CQ) on the sepsis-induced lethality of HIPK2-overexpressing mice ( n = 30 mice per group); + p < 0.05 compared with the CLP plus Ad-vector group in the absence of CQ and @ p < 0.05 compared with the CLP plus Ad-HIPK2 group in the absence of CQ. In this section, CLP ( n = 30) was performed on the fifth day after the adenovirus injection, and CQ (50 mg/kg) was administered 1 h prior to CLP. i Effect of HIPK2 overexpression on LPS (0.5 μg/mL)-induced apoptosis of primary hepatocytes, as analysed by flow cytometry. j Percentage of apoptotic cells induced by LPS; data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the Ad-vector group, + p < 0.05 compared with the LPS plus Ad-vector group

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Plasmid Preparation, Expressing, Injection, Over Expression, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a Effect of HIPK2 overexpression on Beclin-1 and Atg12-5 levels, as analysed by western blotting; CLP ( n = 30) was performed on the fourth day after the adenovirus injection. Data are presented as means ± SEM and are representative of three separate experiments; # p < 0.05 compared with the sham plus Ad-vector group (control), + p < 0.05 compared with the CLP plus Ad-vector group. b Effect of HIPK2 overexpression on Atg3 and Atg7 levels, as analysed by western blotting. Data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the sham plus Ad-vector group, + p < 0.05 compared with the CLP plus Ad-vector group

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Over Expression, Western Blot, Injection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a , b Effects of Ad-HIPK2 on levels of the LAMP-2, Rab7, and cathepsin B proteins in the liver, as analysed by western blotting ( a ) and the results of the corresponding semi-quantitative analysis of levels of the HIPK2 protein based on the optical density measured using ImageJ software ( b ); data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the sham plus Ad-vector group (control), + p < 0.05 compared with the CLP plus Ad-vector group. c , d Effects of Ad-HIPK2 on levels of the LC3 and LAMP-2 proteins in the liver, as determined by IHC ( c ) and the results of the corresponding semi-quantitative analysis of levels of the HIPK2 protein based on the optical density measured using ImageJ software ( d ); and data are presented as means ± SEM and are representative of six microscopic fields per group, # p < 0.05 compared with the sham plus Ad-vector group (control), + p < 0.05 compared with the CLP plus Ad-vector group. e Images of H & E-stained liver sections captured at a magnification of 200 × (six microscopic fields per group)

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Western Blot, Software, Plasmid Preparation, Staining

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a , b , c Effect of Ad-HIPK2 on p-mTOR, p-4E-BP1, and p-p70S6K levels, as determined by western blotting ( a , b ) and effect of Ad-HIPK2 on the activation of the calpain system, as analysed by western blotting ( a , c ). The results of the corresponding semi-quantitative analysis of protein levels was based on the optical density measured using ImageJ software. Data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the sham plus Ad-vector group (control), + p < 0.05 compared with the CLP plus Ad-vector group. d , e , f Effect of Ad-shHIPK2 on p-mTOR, p-4E-BP1 and p-p70S6K levels after the administration of rapamycin (1 mg/kg per day) for 24 h, as determined by western blotting ( d , e ) and effect of Ad-shHIPK2 on the activation of the calpain system in the presence of rapamycin, as analysed by western blotting ( d , f ). Results of the corresponding semi-quantitative analysis of protein levels based on the optical density measured using ImageJ software. Data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the sham plus Ad-NC group, + p < 0.05 compared with the CLP plus Ad-NC group, & p < 0.05 compared with the sham plus Ad-shHIPK2 group

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Western Blot, Activation Assay, Software, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsis-mediated liver injury by restoring autophagy

doi: 10.1038/s41419-018-0838-9

Figure Lengend Snippet: a Interactions of HIPK2 with calpain 1 or calmodulin in primary hepatocytes were analysed by immunoprecipitation after an incubation with 0.5 μg/mL LPS for 12 h. b HIPK2 overexpression downregulated cytosolic Ca 2+ concentrations after an incubation with 0.5 μg/mL LPS for 12 h, as determined by flow cytometry. c , d HIPK2 expression was upregulated by treatments with 5 μM resveratrol, 30 μM aspirin, 10 μM vitamin E, and 15 μM ursolic acid for another 16 h after the LPS treatment, as analysed by western blotting ( c ) and Q-PCR ( d ) and the corresponding semi-quantitative analysis of protein levels was based on the optical density measured using ImageJ software; data are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the LPS plus placebo (control) group. e , f Effects of the indicated drugs on the luciferase activity of HIPK2 promoter and 3′-UTR stability; the results are presented as means ± SEM and are representative of three separate experiments, # p < 0.05 compared with the control. g To knockdown HIPK2 expression, the primary hepatocytes were infected with Ad-shHIPK2 (2 × 10 3 plaque-forming units per well, six-well plate). After 12 h of infection, the effects of the indicated drugs on calpain 1-mediated autophagy were determined by western blotting in three experiments

Article Snippet: For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control.

Techniques: Immunoprecipitation, Incubation, Over Expression, Flow Cytometry, Expressing, Western Blot, Software, Luciferase, Activity Assay, Infection