Journal: Cell Death & Disease

Article Title: IMP2/p62 induces genomic instability and an aggressive hepatocellular carcinoma phenotype

doi: 10.1038/cddis.2015.241

Figure Lengend Snippet: p62 promotes DLK1-RAC1-driven ROS generation. ( a and b ) Levels of RAC1 mRNA presented as mean±S.E.M. ( a ) and activated RAC1 protein levels determined by pull-down assay ( b ) in HepG2 cells after treatment with 1 μ g/ml DLK1 protein ( n =3 in duplicate). ( b ) Representative pull-down assay with activated RAC1 (aRAC1) and total RAC1 (tRAC1) is shown. X-fold signal intensities of 5-min treatment with DLK1 were normalized to untreated control (co). ( c ) ROS levels: representative experiment (quintuplicates) of HepG2 cells treated with 0.5 or 1 μ g/ml DLK1 or H 2 O 2 as positive control for 0–30 min (upper part). Data are normalized to untreated HepG2 cells. ROS levels in HepG2 cells treated with either DLK1 or RAC1 inhibitor NSC23766 alone or in combination (lower part). Untreated HepG2 cells served as control. H 2 O 2 -induced ROS formation was set to 100% ( n =2, quintuplicate). Data are presented as mean±S.E.M. ( d ) RAC1 expression in HepG2 cells overexpressing (left) p62 sense plasmid (p62) compared with antisense-plasmid (co-v), untreated control (co), and siRNA knockdown (right) of p62 (si p62) compared with random siRNA (si co; n =3 triplicate/quadruplicate). Data show mean±S.E.M. Western blot knockdown/overexpression control was densitometrically quantified ( n =4 triplicate/quadruplicate; upper part). ( e ) RAC1 expression in human HCC (GSE14520) normalized to the mean of normal samples. ( f ) Overview of p62-promoted DLK1-RAC1-induced genomic instability. DLK1-overexpressing cells with stem-cell-like features secrete DLK1 protein, which activates RAC1 in a paracrine manner, in turn leading to ROS generation via NADPH oxidase. Elevated ROS levels finally result in genomic instability

Article Snippet: Knockdown and overexpression experiments for p62 in HepG2 were performed as previously described. p62 sense and antisense constructs are available at Addgene (#42174 and #42175, Cambridge, MA, USA).

Techniques: Pull Down Assay, Control, Positive Control, Expressing, Plasmid Preparation, Knockdown, Western Blot, Over Expression

Journal:

Article Title: ATPase Class I Type 8B Member 1 and Protein Kinase C-? Induce the Expression of the Canalicular Bile Salt Export Pump in Human Hepatocytes

doi: 10.1203/PDR.0b013e3181c2df16

Figure Lengend Snippet: BSEP Promoter Analysis in HepG2 cells. HepG2 cells were cultured in 0.5% charcoal treated fetal calf serum that was supplemented with 100 μM chenodeoxycholic acid (CDCA) or 10 μM GW 4064 as FXR ligands. FIC1 and/or FXR were silenced as indicated or PKCζ was inhibited using a pseudosubstrate. Human BSEP promoter activity was assessed using a luciferase reporter. Bars represent the mean of 9 measurements in three separate experiments. Error bars are standard deviations. The y-axis is a logarithmic scale with BSEP luciferase reporter activity reported relative to a transfection efficiency control Renilla luciferase. (Inset - western analysis of FIC1 silencing in HepG2 cells). = 0.5% charcoal treated fetal calf serum, = 100 μM CDCA, = 10 μM GW4064.

Article Snippet: The FIC1 anti-sense plasmid construct pRNA-siFIC1 (siFIC1) and an anti-sense control plasmid construct pRNA-siScr (siScr) were purchased from GenScript Corporation (Piscataway, New Jersey) ( 10 ).

Techniques: Cell Culture, Activity Assay, Luciferase, Transfection, Control, Western Blot

Journal:

Article Title: ATPase Class I Type 8B Member 1 and Protein Kinase C-? Induce the Expression of the Canalicular Bile Salt Export Pump in Human Hepatocytes

doi: 10.1203/PDR.0b013e3181c2df16

Figure Lengend Snippet: Western Blot Analysis of the Effect of FIC1 Silencing on BSEP Protein Expression. Human hepatocytes were treated with siFIC1 or siscrambled, membrane proteins were prepared, and 20 μg of those proteins were probed with antibodies directed against FIC1, BSEP, and Na+/K+-ATPase (membrane loading control). FIC1 silencing is efficient (siscrambled 43.1 + 13.7 vs siFIC1 1.4 + 1.5 densitometry units, n =3, p< .001) and associated with a reduction in BSEP protein expression (siscrambled 44.2 + 6.9 vs siFIC1 3.8 + 0.6, densitometry units, n= 3, p <.001). Equivalent loading is indicated by the signal for Na+/K+-ATPase. This blot is representative of three separate experiments with different human hepatocyte preparations.

Article Snippet: The FIC1 anti-sense plasmid construct pRNA-siFIC1 (siFIC1) and an anti-sense control plasmid construct pRNA-siScr (siScr) were purchased from GenScript Corporation (Piscataway, New Jersey) ( 10 ).

Techniques: Western Blot, Expressing, Membrane, Control

Journal:

Article Title: ATPase Class I Type 8B Member 1 and Protein Kinase C-? Induce the Expression of the Canalicular Bile Salt Export Pump in Human Hepatocytes

doi: 10.1203/PDR.0b013e3181c2df16

Figure Lengend Snippet: Western Blot Analysis of the Effect of FIC1 Silencing on FXR. A. Human hepatocytes were treated with siFIC1 or siscrambled, 50 μg of cytoplasmic and 2.5 μg of nuclear proteins were prepared and probed with antibodies directed against FXR, SHP and the loading controls actin (cytoplasmic) and Histone H1 (nuclear). FIC1 silencing leads to an overall reduction in FXR and SHP protein abundance with a redistribution of the FXR from a predominant nuclear to cytoplasmic localization in the FIC1 siRNA treated cells. B. FXR was immunoprecipitated from the same protein extracts and then probed with general antibodies against phosphorylated (P-) threonine (Thr), serine (Ser), or tyrosine (Tyr). In the scrambled antisense control treated cells, FXR is phosphorylated on Thr and Ser but not Tyr. Phosphorylation is not detectable after siFIC1 treatment. In A and B densitometry of specific bands is shown below the band.

Article Snippet: The FIC1 anti-sense plasmid construct pRNA-siFIC1 (siFIC1) and an anti-sense control plasmid construct pRNA-siScr (siScr) were purchased from GenScript Corporation (Piscataway, New Jersey) ( 10 ).

Techniques: Western Blot, Quantitative Proteomics, Immunoprecipitation, Control, Phospho-proteomics