Journal: PLoS Biology

Article Title: Non-proteolytic ubiquitin modification of PPARγ by Smurf1 protects the liver from steatosis

doi: 10.1371/journal.pbio.3000091

Figure Lengend Snippet: (A) Western blots showing that knockdown of Smurf1 but not Smurf2 in Hep3B and AML12 cells increased PPARγ protein level. (B) qRT-PCR analyses showing that knockdown of Smurf1 but not Smurf2 increased Ppar γ mRNA level in AML12 cells. (C) qRT-PCR analyses showing that knockdown of Smurf1 in AML12 cells increased expression of Fabp1 , Cd36 , Acacb , and Apoc3 in a PPARγ-dependent manner. (D) Fatty acid uptake in AML12 cells as measured by 3 H-palmitate incorporation ( n = 3). (E) Lipid synthesis in AML12 cells as measured by incorporation of 3 H-acetate into lipid ( n = 3). (F) In vivo fatty acid uptake after intraperitoneal injection of BODIPY-FL-C16. The BODIPY-FL-C16 accumulation in the liver, epididymal WAT, and skeletal muscle was normalized to tissue weight ( n = 8 per group). (G) Lipogenesis in primary hepatocytes as measured by the incorporation of 3 H-acetate into lipid ( n = 6 per group). Data are presented as mean ± SD; statistical significance of difference is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Original raw data can be found in . BODIPY-FL-C16, 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza- s -Indacene-3-Hexadecanoic Acid; eWAT, epididymal WAT; HSC70, heat shock cognate 71 kDa protein; NS, non-silencing control; PPAR, peroxisome proliferator-activated receptor; qRT-PCR, quantitative real-time PCR; RFU, relative fluorescence units; Rxr, retinoid x receptor; SF1KO, Smurf1 KO; siNS, non-silencing control siRNA; Smurf, Smad ubiquitin regulatory factor; WAT, white adipose tissue; WT, wild-type.

Article Snippet: AML12 cells (ATCC CRL-2254) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone.

Techniques: Western Blot, Knockdown, Quantitative RT-PCR, Expressing, In Vivo, Injection, Control, Real-time Polymerase Chain Reaction, Fluorescence, Ubiquitin Proteomics

Journal: PLoS Biology

Article Title: Non-proteolytic ubiquitin modification of PPARγ by Smurf1 protects the liver from steatosis

doi: 10.1371/journal.pbio.3000091

Figure Lengend Snippet: (A) Co-immunoprecipitation showing interaction between endogenous Smurf1 and PPARγ in AML12 cells. *nonspecific band. (B) PY motif in PPARγ contributes to the interaction between Smurf1 and PPARγ. Myc-tagged Smurf1, Flag-tagged PPARγ2, and its ΔPY mutant were transfected into AML12 cells as indicated. WCL were immunoprecipitated with Flag-M2 beads and followed by western blot analyses. (C) Smurf1 but not Smurf2 promotes polyubiquitination of PPARγ1 and PPARγ2. Flag-PPARγ were immunoprecipitated from transfected AML12 and resolved by SDS-PAGE. Western blot analyses were carried out to detect HA-ubiquitin (top) and Flag-PPARγ1 or -γ2 (second panel) in the precipitates. The levels of total HA-Ub, Flag-PPARγ, Myc-Smurfs, and endogenous Hsc70 (loading control) in the WCL were also analyzed and are shown in the bottom panels. *nonspecific band. (D) E3 ligase activity of Smurf1 is required for Smurf1-mediated polyubiquitination of PPARγ. Flag-PPARγ2 and WT Myc-Smurf1 and its mutant Myc-Smurf1(CA) were transfected into the Smurf1KO MEFs along with HA-Ub. Ubiquitination of PPARγ2 was analyzed by western blot after Flag-M2 immunoprecipitation, as in C. (E) In vitro ubiquitination assay using recombinant proteins showing that PPARγ is a direct substrate of Smurf1-mediated polyubiquitination. (F) Smurf1 induces K63-linked polyubiquitination of PPARγ. Purified ubiquitin with no lysine residue (K0) or with single lysine residue at indicated position was used in the in vitro ubiquitination assay. E3, ubiquitin ligase; HA, human influenza hemagglutinin; Hsc70, heat shock cognate 71 kDa protein; IB, immunoblot; IP, immunoprecipitation; K, lysine; KO, knockout; MEF, mouse embryonic fibroblast; PPAR, peroxisome proliferator-activated receptor; PY, PPxY; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Smurf, Smad ubiquitin regulatory factor; Ub, ubiquitin; WCL, whole cell lysate; WT, wild-type.

Article Snippet: AML12 cells (ATCC CRL-2254) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone.

Techniques: Immunoprecipitation, Mutagenesis, Transfection, Western Blot, SDS Page, Ubiquitin Proteomics, Control, Activity Assay, In Vitro, Recombinant, Purification, Residue, Knock-Out, Polyacrylamide Gel Electrophoresis

Journal: PLoS Biology

Article Title: Non-proteolytic ubiquitin modification of PPARγ by Smurf1 protects the liver from steatosis

doi: 10.1371/journal.pbio.3000091

Figure Lengend Snippet: (A) Smurf1 inhibits PPARγ-induced transcriptional activity in AML12 cells. Relative luciferease activities were measured 1 day after transfection. Data are presented as mean ± SD; statistical significance of differences is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001. Expression of transfected Smurf1 and PPARγ in these cells is shown at right. (B) E3 ligase activity of Smurf1 is required for its inhibition of PPARγ transcriptional activity. Luciferase activities were measured and showed as above. Expression of transfected Smurf1 and PPARγ in these cells are shown at right. (C) ChIP analyses of PPARγ binding to its own or Fabp1 promoter in AML12 cells after transfecting the plasmids as indicated. (D) ChIP analyses of PPARγ binding to its own or Fabp1 promoter in liver tissues from WT and Smurf1KO mice ( n = 8 per group). Data are presented as mean ± SD; statistical significance of difference is indicated by * p < 0.05, ** p < 0.01, *** p< 0.001. Original raw data can be found in . ChIP, chromatin immunoprecipitation; E3, ubiquitin ligase; Hsc70, heat shock cognate 71 kDa protein; IgG, Immunoglobulin G; KO, knockout; PPAR, peroxisome proliferator-activated receptor; PPRE-Luc, PPAR response element-luciferase reporter; SF1KO, Smurf1 KO; Smurf, Smad ubiquitin regulatory factor; WT, wild-type.

Article Snippet: AML12 cells (ATCC CRL-2254) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone.

Techniques: Activity Assay, Transfection, Expressing, Inhibition, Luciferase, Binding Assay, Chromatin Immunoprecipitation, Ubiquitin Proteomics, Knock-Out

Journal: Cells

Article Title: Ursodeoxycholic Acid Regulates Hepatic Energy Homeostasis and White Adipose Tissue Macrophages Polarization in Leptin-Deficiency Obese Mice

doi: 10.3390/cells8030253

Figure Lengend Snippet: Ursodeoxycholic acid (UDCA) alleviates high free fatty acid (HFFA)-induced hepatocyte lipogenesis, reactive oxygen species (ROS) production, and mitochondrial dysfunction in AML12 cells. AML12 cells were treated with 1 mM HFFA with 10, 30, 100 μM UDCA. ( A ) Lipid accumulation display using Oil Red O stain (red). ROS levels were measured using DCFH-DA (green) stain. Images of AML12 cells stained with Mito Tracker for mitochondria (red). qRT-PCR analysis of ( B ) Complex I, II, III, IV, and V mRNA expression in AML12 cells. Relative mRNA expression was normalized to Gapdh and then normalized to the controls. ( C ) Immunofluorescence analysis of SREBP1c (green), CD36 (red), NF-κB (green), and FXR (green) expression, and DAPI (blue) for nuclear. Scale bar, 25 μm. qRT-PCR analysis of ( D ) Srebp-1c, Fas , and Scd-1 mRNA expression in AML12 cells. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student’s t -test (* p ≤ 0.05; 0 mM HFFA vs. 1 mM HFFA. # p ≤ 0.05; 1 mM HFFA vs. 1 mM HFFA+ 100 μM UDCA). UDCA, ursodeoxycholic acid; HFFA, high free fatty acid; ROS, reactive oxygen species; SREBP-1c, sterol regulatory element-binding protein-1c; CD36, cluster of differentiation 36; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; FXR, farnesoid X receptor; Fas , fatty acid synthase; Scd-1, stearoyl-CoA desaturase-1; qRT-PCR, quantitative real-time polymerase chain reaction; Gapdh , glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Alpha mouse liver 12 (AML12) cell line (passages 7–10, obtained from Bioresource Collection and Research Centre, Taiwan) was grown at 37 °C in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F12), 10% fetal bovine serum (FBS), 2 mmol/L l -Glutamine and 100 μg/mL penicillin and streptomycin (both from Gibco, AntiSel, Greece) at 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMAO-Activated Hepatocyte-Derived Exosomes Impair Angiogenesis via Repressing CXCR4

doi: 10.3389/fcell.2021.804049

Figure Lengend Snippet: Isolation and characterization of exosomes from hepatocyte culture supernatant. (A) Nanovesicles with diameters around 100 nm were isolated and purified from the AML12 cell culture supernatant, which possessed the characteristic size range of exosomes (Exos) under electron microscopes. Bar: 200 nm. (B) The size distribution of the Exos showed no significant difference between trimethylamine-N-oxide (TMAO)-free group (Control-Exos) and TMAO-Exos. (C) Exosomal markers of CD9 and TSG101 were enriched in Exos groups, and the negative markers of calnexin were detected only in whole cell lysate. (D) TMAO was undetectable in Control-Exos, but a small quantity of TMAO remained in TMAO-Exos. Data were expressed as mean ± standard error of the mean (SEM). n = 3, independent t -test was performed for comparisons; ** p < 0.01 versus Control-Exos. (E) Exosomes were labelled with DiI and co-cultured with human aortic endothelial cells (HAECs) for 24 h, and it was shown that DiI-labeled Exos could be taken up by cells (×400 magnification).

Article Snippet: AML12 cells (iCell Bioscience Inc, Shanghai, China) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing Exos-depleted serum (ViVaCell, Shanghai, China), and treated with TMAO (Tokyo Chemical Industry, Japan) at a physiological concentration of 50 μmol/l for 48 h. Exos were isolated and purified from the culture supernatant using differential centrifugation and then resuspended in phosphate-buffered saline (PBS), as described in our previous study ( ).

Techniques: Isolation, Purification, Cell Culture, Control, Labeling