Journal: Journal of Clinical and Translational Hepatology

Article Title: Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling

doi: 10.14218/JCTH.2023.00562

Figure Lengend Snippet: (A) GRIM19 loss induces abnormal ROS release in vitro . Intracellular ROS and mitochondrial ROS (mROS) were detected by flow cytometry in AML12 cells. (B–D) GRIM19 loss induces aberrant oxidative stress in vitro . DNA damage marker 8-oxodeoxyguanosine (8-OHdG) was detected by immunofluorescence (IF) staining in GRIM19-deficient AML12 cells (B). Oxidative stress was evaluated by analyzing intracellular ATP content (C), GSH/GSSG ratio, and NADP+/NADPH ratio (D) in GRIM19-deficient AML12 cells. (E, F) GRIM19 loss induces abnormal ROS release in vivo . Intracellular ROS (E) and mROS (F) were detected by IF staining in fresh frozen liver tissues from two-year-old mice. (G) GRIM19 loss causes oxidative damage in vivo . DNA damage 8-OHdG was determined by IF staining in fresh frozen liver tissues from two-year-old mice. Representative images are shown. DAPI was used to stain the nuclei. Mean fluorescent intensity (MFI) was used to quantify protein expression in IF staining. Data are expressed as mean±SD. Scale bar: 50 µm. ** p <0.01, *** p <0.001 between the indicated groups determined by unpaired student’s t-test . AML-12, alpha mouse liver 12; NC, negative control; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Murine hepatocyte cells AML12 (Procell, Wuhan, China) were maintained in DMEM/F12 medium supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA), 1× insulin-transferrin-selenium media supplement (Procell, Wuhan, China), 40 ng/mL dexamethasone (Procell, Wuhan, China), streptomycin (100 ug/mL), and penicillin (100 U/mL) at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: In Vitro, Flow Cytometry, Marker, Immunofluorescence, Staining, In Vivo, Expressing, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling

doi: 10.14218/JCTH.2023.00562

Figure Lengend Snippet: (A–F) GRIM19 loss promotes NF-кB/p65 activation in vitro and in vivo. Total p65, p-p65, and NF-кB downstream targets IL6, TNFα, VEGF, VCAM1, and ICAM1 were analyzed by western blotting in GRIM19-deficient AML12 cells (A) and liver tissues (B). NF-кB-regulatory proteins pIKKα/β, IKKα/β, pIкBα, and IкBα were detected by western blotting in GRIM19-deficient AML12 cells and liver tissues (C). NF-кB p65 levels in nuclear or cytoplasmic extractions were analyzed by western blotting in GRIM19-deficient AML12 cells (D). p65 and p-p65 co-expression was detected by dual immunofluorescence (IF) staining in GRIM19-deficient AML12 cells (E) and liver tissues (F). (G) NF-кB inhibition reverses GRIM19 loss-driven NF-кB/p65 activation in vitro . GRIM19-deficient AML12 cells were treated with NF-кB inhibitor PDTC (0, 5, 10 µM) for 16 h. The expression of p65, p-p65, and NF-кB downstream targets were determined by western blotting. (H, I) Reactive oxygen species (ROS) scavenger abrogates GRIM19 loss-driven NF-кB/p65 activation in vitro . GRIM19-deficient AML12 cells were treated with NAC (0, 5, 10 mM) for 16 h, then p65, p-p65, and NF-кB downstream targets were detected by western blotting (H). NF-кB-regulatory proteins pIKKα/β, IKKα/β, pIкBα, and IкBα were detected by western blotting in GRIM19-deficient AML12 cells after NAC treatment (0, 5, 10 mM) for 16 h (I). β-actin was used as a loading control. DAPI was used to stain the nuclei. Mean fluorescent intensity (MFI) was used to quantify the expression of proteins in IF staining. Data are expressed as mean±SD. Scale bars: 50 µm. * p <0.05, ** p <0.01, *** p <0.001 between the indicated groups determined by unpaired student’s t-test . AML-12, alpha mouse liver 12; NC, negative control; NAC, N-acetylcysteine; PDTC, ammonium pyrrolidinedithiocarbamate; IL-6, interleukin 6; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intercellular adhesion molecular 1; VEGF, vascular endothelial growth factor; TNF, tumour necrosis factor; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Murine hepatocyte cells AML12 (Procell, Wuhan, China) were maintained in DMEM/F12 medium supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA), 1× insulin-transferrin-selenium media supplement (Procell, Wuhan, China), 40 ng/mL dexamethasone (Procell, Wuhan, China), streptomycin (100 ug/mL), and penicillin (100 U/mL) at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Activation Assay, In Vitro, In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Inhibition, Control, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling

doi: 10.14218/JCTH.2023.00562

Figure Lengend Snippet: (A) GRIM19 loss increases IL1β and IL33 levels. IL1β and IL33 cytokines were detected by flow cytometry in GRIM19-deficient AML12 cells. (B) GRIM19 loss triggers NLRP3 inflammasome activation in vitro . NLRP3 inflammasome complex, as well as IL1β and IL33, were detected by western blotting in GRIM19-deficient AML12 cells. (C) ROS inhibition attenuates GRIM19 loss-induced NLRP3 inflammasome activation in vitro . GRIM19-deficient AML12 cells were treated with NAC (0, 5, 10 mM) for 16 h. NLRP3 inflammasome, IL1β and IL33 were detected by western blotting. (D) NF-кB blockage decreases GRIM19 loss-triggered NLRP3 inflammasome activation in vitro . GRIM19-deficient AML12 cells were treated with NF-кB inhibitor PDTC (0, 5, 10 µM) for 16 h. NLRP3 inflammasome, IL1β, and IL33 levels were detected by western blotting. (E) NLRP3 inhibition reduces GRIM19 loss-induced IL1β and IL33 expression in vitro . GRIM19-deficient AMl12 cells were treated with NLRP3 inhibitor MCC950 (0, 2.5, 5 µM) for 16 h. NLRP3 inflammasome, IL1β, and IL33 were detected by western blotting. (F) Caspase1 repression attenuates GRIM19 loss-triggered IL1β and IL33 expression in vitro . GRIM19-deficient AML12 cells were treated with Caspase1 inhibitor VX765 (0, 20, 40 µM) for 24h. Caspase1, IL1β, and IL33 expressions were determined by western blotting. β-actin was used as a loading control. Data are presented as mean±SD of three independent experiments. Representative images are shown. IL, interleukin; NAC, N-acetylcysteine; PDTC, ammonium pyrrolidine dithiocarbamate; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; NC, negative control.

Article Snippet: Murine hepatocyte cells AML12 (Procell, Wuhan, China) were maintained in DMEM/F12 medium supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA), 1× insulin-transferrin-selenium media supplement (Procell, Wuhan, China), 40 ng/mL dexamethasone (Procell, Wuhan, China), streptomycin (100 ug/mL), and penicillin (100 U/mL) at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Flow Cytometry, Activation Assay, In Vitro, Western Blot, Inhibition, Expressing, Control, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A–E) APOA4 , PCSK9 , HMGCR, ABCG5 , and ABCG8 mRNA expression levels in TMAO-treated and control AML12 cells (n = 3 per group). (F) Immunofluorescence staining for APOA4, PCSK9, HMGCR, ABCG5, and ABCG8 in cells from each group. ** p < 0.01, *** p < 0.001. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.

Article Snippet: The murine normal hepatocyte line AML12 was acquired from Cyagen Biosciences Inc. (Shanghai, China) and cultured using AML12 cell-specific culture medium (Procell, Wuhan).

Techniques: Expressing, Control, Immunofluorescence, Staining, Binding Assay