Journal: Nature Communications

Article Title: The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes

doi: 10.1038/s41467-022-28641-w

Figure Lengend Snippet: a Records for the liver weight (upper) and the ratio of liver weight/body weight (lower) (%) of the Flox and THKO mice at the last week of HFD treatment ( n = 10 mice per group) (** P < 0.01 vs. Flox HFD groups). b Representative pictures for liver appearance and transmission electron microscope (TEM)-indicated histological changes of the liver in Flox and THKO mice after NCD or HFD feeding for 16 weeks (Scale bar, 10 μm for upper image, 2 μm for the lower image; n = 10 images per group for each group) (** P < 0.01 vs. Flox HFD groups). ( c ) Liver lipid contents including triglyceride (TG), total cholesterol (TC) and non-esterified fatty acids (NEFA) (upper), and serum alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) levels (lower) of the Flox and THKO mice after HFD treatment for 16 weeks ( n = 10 mice per group) (** P < 0.01 vs. Flox HFD groups). d Representative pictures of H&E-stained (upper) and Oil-red O-stained (lower) pathological section of the liver from the 16-week HFD-fed Flox and THKO mice (magnification, ×100; n = 10 images per group for each staining) (** P < 0.01 vs. Flox HFD groups). e qPCR analysis of the relative mRNA expression of genes associated with fatty acid uptake, synthesis, and β-oxidation in Flox and THKO mice after 16-week HFD feeding ( n = 10 liver samples per group) (** P < 0.01 vs. Flox HFD groups). f Records for the liver weight (upper) and the ratio of liver weight/body weight (lower) (%) of the non-transgenic (NTG) mice and hepatocyte Trim31 transgenic (THTG) mice at the last week of HFD treatment ( n = 10 mice per group) (** P < 0.01 vs. NTG HFD groups). g Representative pictures for liver appearance and TEM-indicated histological changes of the liver in NTG and THTG mice after 16-week NCD or HFD feeding (Scale bar, 10 μm for upper image, 2 μm for the lower image; n = 10 images per group for each group) (** P < 0.01 vs. NTG HFD groups). h Liver lipid contents including TG, TC, and NEFA (upper), and serum ALT, AST, and AKP levels (lower) of the NTG and THTG mice after HFD treatment for 16 weeks ( n = 10 mice per group) (** P < 0.01 vs. NTG HFD groups). i Representative pictures of H&E-stained (upper) and Oil-red O-stained (lower) patholog i cal section of the liver from the 16-week HFD-fed NTG and THTG mice (magnification, ×100; n = 10 images per group for each staining) (** P < 0.01 vs. NTG HFD groups). j qPCR analysis of the relative mRNA expression of genes associated with fatty acid uptake, synthesis, and β-oxidation in NTG and THTG mice after 16-week HFD feeding ( n = 10 liver samples per group) (** P < 0.01 vs. NTG HFD groups). k Representative pictures of Oil red O staining of primary hepatocytes that were transfected with AdTrim31 or AdshTrim31 and/or treated with corresponding controls or PA for 10 h (magnification, ×200; n = 10 images per group for each staining) (** P < 0.01 vs. AdshRNA palmitate groups (upper) and AdGFP palmitate groups (lower)). Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. Significance determined by Student’s two-tailed t test analysis.

Article Snippet: The concentration of serum alanine transaminase (ALT) (#MAK052, Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, Sigma-Aldrich), alkaline phosphatase (AKP) (#ab83369, Abcam), serum insulin (#ab277390, Abcam) and hepatic triglyceride (TG) (#MAK266, Sigma-Aldrich), total cholesterol (TC) (#ab65359, Abcam) and non-esterified free fatty acids (NEFA) (#E-BC-K014, Elabscience, Inc., Houston, USA) were detected using commercially-available detection kits in the indicated groups according to the product specification.

Techniques: Transmission Assay, Microscopy, Staining, Expressing, Transgenic Assay, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes

doi: 10.1038/s41467-022-28641-w

Figure Lengend Snippet: Preconditioned liver-specific Trim31 deletion (THKO) mice with an 8-week HFD treatment as donors were transduced (THKO)(LV−). The hepatocytes isolated from the (THKO)(LV−) group mice were transduced with a lentivirus-loaded full-length Trim31 sequence. The corresponding blank vector was transduced as controls. Then the additional HFD-fed THKO mice as recipient were injected with transduced hepatocytes via the portal vein. The HFD-fed transplanted (THKO)(LV+) mice were harvested for further experimental detection. a The strategy diagram for ex vivo gene therapy used in the current study. b Records for the liver weight, body weight, and the ratio of liver weight/body weight (%) of the HFD-fed recipient (THKO)(LV−) and (THKO)(LV+) mice at the last week of HFD treatment ( n = 25 mice per group) (** P < 0.01 vs. HFD-fed recipient (THKO)(LV−) groups). c Representative pictures for liver appearance and Oil red O staining, H&E staining, and PAS staining-indicated liver histopathologic changes of the HFD-fed recipient (THKO)(LV−) and (THKO)(LV+) mice after ex vivo experiment (magnification, ×100; n = 10 images per group for each staining). d Liver function markers ALT and AST levels were detected in transduced recipient mice ( n = 25 mice per group) (** P < 0.01 vs. HFD-fed recipient (THKO)(LV-) groups). e , f Records for the glucose tolerance test (GTT) ( e ) and insulin tolerance test (ITT) ( f ), and the corresponding fasting insulin levels and HOMA-IR index in the HFD-fed recipient (THKO)(LV−) and (THKO)(LV+) mice after transplantation ( n = 25 mice per group) (** P < 0.01 vs. HFD-fed recipient (THKO)(LV−) groups). g Liver lipid contents including TG, TC, and NEFA of the transduced mice ( n = 25 mice per group) (** P < 0.01 vs. HFD-fed recipient (THKO)(LV−) groups). h qPCR analysis of the relative mRNA expression of genes associated with fatty acid uptake, synthesis, and β-oxidation in the HFD-fed recipient (THKO)(LV−) and (THKO)(LV+) mice after transplantation ( n = 10 liver samples per group) (** P < 0.01 vs. HFD-fed recipient (THKO)(LV-) groups). i , j Representative immunoblotting bands for expression alterations of total amounts and phosphorylated forms of critical indicators involved in insulin signaling ( i ), including IRS1, p-IRS1(Ser307), p-IRS1(Tyr608), AKT, and p-AKT, and Rhbdf2–MAP3K7 axis and p-NF-κB pathway ( j ) in the liver of HFD-fed recipient (THKO)(LV−) and (THKO)(LV+) mice ( n = 4 per experiment) (** P < 0.01 vs. HFD-fed recipient (THKO)(LV−) groups). The GAPDH was used as a loading control. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. Significance determined by Student’s two-tailed t test analysis.

Article Snippet: The concentration of serum alanine transaminase (ALT) (#MAK052, Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, Sigma-Aldrich), alkaline phosphatase (AKP) (#ab83369, Abcam), serum insulin (#ab277390, Abcam) and hepatic triglyceride (TG) (#MAK266, Sigma-Aldrich), total cholesterol (TC) (#ab65359, Abcam) and non-esterified free fatty acids (NEFA) (#E-BC-K014, Elabscience, Inc., Houston, USA) were detected using commercially-available detection kits in the indicated groups according to the product specification.

Techniques: Isolation, Transduction, Sequencing, Plasmid Preparation, Injection, Ex Vivo, Staining, Transplantation Assay, Expressing, Western Blot, Control, Two Tailed Test

Journal: Nature Communications

Article Title: The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes

doi: 10.1038/s41467-022-28641-w

Figure Lengend Snippet: a Representative immunoblotting bands of Trim31, Rhbdf2, and corresponding downstream event indicator levels of total and phosphorylated ADAM17, MAP3K7, p-MAP3K7, NF-κB, p-NF-κB, IκBα, p-IκBα, JNK1/2, and p-JNK1/2 in liver samples isolated from the indicated mice fed a HFD for 16 weeks. b , c Records for body weight, liver weight, the ratio of liver weight/body weight (%) ( b ), and fasting blood glucose levels, fasting insulin levels, and HOMA-IR index ( c ) in a 16-week HFD-fed indicated mice ( n = 10 mice per group) (** P < 0.01 vs. Flox HFD groups, ## P < 0.01 vs. THKO HFD groups). d Representative pictures for Oil red O staining and H&E staining-indicated liver histopathologic changes of a 16-week HFD-fed indicated mice (magnification, ×100; n = 10 images per group for each staining) (** P < 0.01 vs. Flox HFD groups, ## P < 0.01 vs. THKO HFD groups). e Liver lipid contents including TG, TC, and NEFA of the indicated mice after a 16-week HFD ingestion ( n = 10 mice per group) (** P < 0.01 vs. Flox HFD groups, ## P < 0.01 vs. THKO HFD groups). f qPCR analysis of the relative mRNA expression of genes in the livers associated with inflammation signaling in 16-week HFD-fed mice ( n = 10 liver samples per group) (* P < 0.05 vs. Flox HFD groups, # P < 0.05 vs. THKO HFD groups). g Representative images of Oil red O staining of primary hepatocytes that were transfected with AdshRhbdf2 or AdshTrim31 or co-treated with AdshRhbdf2/AdshTrim31 or PA for 10 h (magnification, ×200; n = 10 images per group for each staining) (** P < 0.01 vs. Flox HFD groups, ## P < 0.01 vs. THKO HFD groups). The relevant experiments presented in this part were performed independently at least three times. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. Significance determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test.

Article Snippet: The concentration of serum alanine transaminase (ALT) (#MAK052, Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, Sigma-Aldrich), alkaline phosphatase (AKP) (#ab83369, Abcam), serum insulin (#ab277390, Abcam) and hepatic triglyceride (TG) (#MAK266, Sigma-Aldrich), total cholesterol (TC) (#ab65359, Abcam) and non-esterified free fatty acids (NEFA) (#E-BC-K014, Elabscience, Inc., Houston, USA) were detected using commercially-available detection kits in the indicated groups according to the product specification.

Techniques: Western Blot, Isolation, Staining, Expressing, Transfection

Journal: Nature Communications

Article Title: The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes

doi: 10.1038/s41467-022-28641-w

Figure Lengend Snippet: a , b Records for liver weight, the ratio of liver weight/body weight ( a ) and time-course changes of body weight in THTG and NTG mice ( b ) after a 16-week HFHF administration ( n = 17 mice per group) (** P < 0.01 vs. NTG HFHF groups, ns., no significant difference). c Liver lipid contents including TG, TC, and NEFA of the indicated mice after 16-week HFHF ingestion ( n = 17 mice per group) (** P < 0.01 vs. NTG HFHF groups). d Representative pictures for Oil red O staining and H&E staining-indicated liver histopathologic changes of a 16-week HFHF-fed THTG and NTG mice (magnification, ×100; n = 10 images per group for each staining) (** P < 0.01 vs. NTG HFHF groups). e qPCR analysis of the relative mRNA expression of genes in the livers associated with fatty acid metabolism after a 16-week HFHF administration ( n = 10 liver samples per group) (** P < 0.01 vs. NTG HFHF groups). f Immunohistochemistry staining of F4/80 analysis showing the histopathologic changes of the liver samples in indicated mice (magnification, ×200; n = 10 images per group for each staining) (** P < 0.01 vs. NTG HFHF groups). g qPCR analysis of the relative mRNA expression of genes in the livers associated with inflammation after a 16-weeks HFHF administration ( n = 10 liver samples per group) (** P < 0.01 vs. NTG HFHF groups). h Representative pictures for Sirius red staining and Masson staining showing the collagen deposition levels of the liver samples in indicated mice (magnification, ×100; n = 10 images per group for each staining). i , j qPCR analysis of the relative mRNA expression of genes in the livers associated with collagen synthesis after a 16-weeks HFHF administration ( i ) ( n = 10 liver samples per group); and hepatic function indicator AST, ALT, and AKP levels in the 16-week HFHF-fed THTG and NTG mice ( j ) ( n = 17 mice per group) (** P < 0.01 vs. NTG HFHF groups). k Schematic diagram showing the molecular mechanism underlying Trim31-mediated Rhbdf2 degradation and protective effects in the development and progression of NASH. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. Significance determined by Student’s two-tailed t test analysis.

Article Snippet: The concentration of serum alanine transaminase (ALT) (#MAK052, Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, Sigma-Aldrich), alkaline phosphatase (AKP) (#ab83369, Abcam), serum insulin (#ab277390, Abcam) and hepatic triglyceride (TG) (#MAK266, Sigma-Aldrich), total cholesterol (TC) (#ab65359, Abcam) and non-esterified free fatty acids (NEFA) (#E-BC-K014, Elabscience, Inc., Houston, USA) were detected using commercially-available detection kits in the indicated groups according to the product specification.

Techniques: Staining, Expressing, Immunohistochemistry, Two Tailed Test