Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model

doi: 10.1007/s00262-015-1690-8

Figure Lengend Snippet: GADD34 deficiency alleviated acute liver injury after DEN treatment. a Representative time courses of three experiments in this study. Intraperitoneal injection (i.p.). Eut, euthanize. b H&E staining of liver sections in the indicated time points after 100 mg/kg DEN treatment (WT mice, n = 22; GADD34−/− mice, n = 22). Black arrows indicate central vein hypertension and necrosis, characterized by hepatocyte vacuolization. Scale bars 200 μm. *P < 0.05, **P < 0.01 versus WT. Histology score represented four samples in each time point and each group (six fields of view for each sample). c Measurement of mouse serum alanine aminotransferase (ALT) activity at the indicated time points after 100 mg/kg DEN treatment. Data from three independent trials are averaged for each time point. *P < 0.05 versus WT. d Representative RT-PCR result of GADD34 (Ppp1r15a) mRNA from WT mice at different times after 100 mg/kg DEN treatment. The statistical data for GADD34 mRNA are normalized to β-actin mRNA in three independent samples. ***P < 0.001, **P < 0.01 versus 0 h. e Real-time PCR analysis of pro-inflammatory cytokine gene Il-6 and oncogene cMyc from WT and GADD34−/− mice at the indicated time points after 100 mg/kg DEN treatment. Data represent mean ± SEM of triplicates for each sample (n = 3). ***P < 0.001 **P < 0.01, *P < 0.05 versus WT; ns no significance versus WT. f Representative analysis of protein expression levels by Western blotting using liver samples from WT and GADD34−/− mice after 100 mg/kg DEN treatment. g Densitometric analysis of band intensities of pp53, pNF-κB, pSTAT3 and pAkt relative to p53, NF-κB, STAT3 and Akt after treatment with DEN. Data represent mean ± SEM of triplicates for samples. ***P < 0.001, **P < 0.01, *P < 0.05 versus WT; ns no significance versus WT

Article Snippet: Anti-F4/80-APC (eBioscience, #17-4801) and anti-IL-6-FITC (eBioscience, #11-7061) were used at 1:500 dilutions and incubated at 4 °C overnight, after which the sections were washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. After rinsed with PBS, the sections were mounted with mounting fluid and visualized under A1Rsi inverted confocal microscopy (Nikon, Tokyo, Japan).

Techniques: Injection, Staining, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model

doi: 10.1007/s00262-015-1690-8

Figure Lengend Snippet: GADD34 deficiency exhibited lower cytokine expression and hepatic compensatory proliferation after chronic DEN treatment. a Real-time PCR analysis of mRNA expression levels of the indicated genes in liver from WT and GADD34−/− mice after chronic DEN treatment. Data represent mean ± SEM of three independent experiments, *P < 0.05, ***P < 0.001 versus WT; ns no significance versus WT. b Representative immunofluorescent confocal microscopic images of tissues co-stained for F4/80 and IL-6 in liver tissues after chronic DEN treatment. Nuclei (4′,6-diamidino-2-phenylindole, DAPI), blue; IL-6, green; F4/80, pink. The red outlined areas are enlarged in top left corners. Scale bars 100 μm. c Statistical analysis of mean fluorescence intensity of IL-6 in Kupffer cells/macrophages. N = 3 with >8 fields of view for each group, *P < 0.05 versus WT, mean ± SEM. no significance versus WT. d Representative protein expression levels of liver samples from WT and GADD34−/− mice after chronic DEN treatment. e Analysis of hepatic compensatory proliferation by staining for Ki67. Black arrows indicate Ki67-positive cells. Scale bars 100 μm. Data represent mean ± SEM of three independent samples with >6 fields of view for each sample (LPF, low-power field). ***P < 0.001 versus WT. ns no significance versus WT

Article Snippet: Anti-F4/80-APC (eBioscience, #17-4801) and anti-IL-6-FITC (eBioscience, #11-7061) were used at 1:500 dilutions and incubated at 4 °C overnight, after which the sections were washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. After rinsed with PBS, the sections were mounted with mounting fluid and visualized under A1Rsi inverted confocal microscopy (Nikon, Tokyo, Japan).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model

doi: 10.1007/s00262-015-1690-8

Figure Lengend Snippet: Schematic summary of this study. The chemical carcinogen diethylnitrosamine (DEN) led to DNA damage that in turn could enhance GADD34 upregulation. GADD34 augmented oncogene activation and the death of DEN-exposed hepatocytes through both pyroptosis and necrosis pathway. This process led to Kupffer cell/macrophage activation and immune cell infiltration that subsequently produced pro-tumorigenic cytokine and ROS, thereby stimulating the compensatory proliferation of surviving, mutant hepatocytes. Finally, abnormal proliferation enhanced HCC progression

Article Snippet: Anti-F4/80-APC (eBioscience, #17-4801) and anti-IL-6-FITC (eBioscience, #11-7061) were used at 1:500 dilutions and incubated at 4 °C overnight, after which the sections were washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. After rinsed with PBS, the sections were mounted with mounting fluid and visualized under A1Rsi inverted confocal microscopy (Nikon, Tokyo, Japan).

Techniques: Activation Assay, Produced, Mutagenesis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model

doi: 10.1007/s00262-015-1690-8

Figure Lengend Snippet: GADD34 deficiency reduced HCC progression through attenuating macrophage infiltration, cytokine level, oncogene expression and hepatic proliferation. a Representative IHC images of non-tumor and tumor areas in liver samples from WT and GADD34−/− mice using anti-F4/80 antibody. The outlined areas are enlarged in the lower panel. Scale bars 50 μm. b Statistical analysis of F4/80-positive cells from different locations in different samples. N = 6 with >8 fields of view for each group, *P < 0.05, ***P < 0.001 versus WT, mean ± SEM. c–e Real-time PCR analysis of indicated genes in non-tumor and tumor areas from WT and GADD34−/− mice. N = 4 for each experiment. *P < 0.05, **P < 0.01 versus WT, mean ± SEM. f Representative IHC results of Ki67 expression in both non-tumor and tumor area. Scale bars 100 μm. g Statistical analysis of Ki67-positive cells in (f). N = 6 with >8 fields of view for each group, **P < 0.01 versus WT, mean ± SEM. no significance versus WT

Article Snippet: Anti-F4/80-APC (eBioscience, #17-4801) and anti-IL-6-FITC (eBioscience, #11-7061) were used at 1:500 dilutions and incubated at 4 °C overnight, after which the sections were washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. After rinsed with PBS, the sections were mounted with mounting fluid and visualized under A1Rsi inverted confocal microscopy (Nikon, Tokyo, Japan).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Endogenous erythropoietin has immunoregulatory functions that limit the expression of autoimmune kidney disease in mice

doi: 10.3389/fimmu.2023.1195662

Figure Lengend Snippet: Downregulating endogenous EPO production increases IL-6 and MCP-1 production in macrophages. Intracellular cytokine production in CD11b + splenic macrophages isolated from B6.MRL/lpr shEPOrtTA POS (n=12) and shEPOrtTA NEG controls (n=10) at day 145 after DOX food initiation (flow cytometry). Mann–Whitney U test; ns, not significant, ** P < 0.01; *** P < 0.001 between B6.MRL/lpr shEPOrtTA POS and shEPOrtTA NEG groups.

Article Snippet: After cell permeabilization using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific), intracellular staining was performed using the following antibodies: FITC-anti-Foxp3, APC-anti-Foxp3 (clone FJK-16S), FITC-anti-IL-6 (clone MP520F3), and FITC-anti-MCP-1 (clone 2H5) (Thermo Fisher Scientific); Pacific Blue-anti-TNF-α (clone MP6-XT22), APC-anti-IL-10 (clone JES5-16E3), PE-Cy7-anti-IL-2 (clone JES6-5H4), BV421-anti-IFN-γ (clone XMG1.2), and PE-Cy7-anti-IL-4 (clone 11B11) (BioLegend).

Techniques: Isolation, Flow Cytometry, MANN-WHITNEY