Journal:

Article Title: The inflammation-induced down-regulation of plasma Fetuin-A (?2HS-Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighbouring binding sites

doi: 10.1093/nar/gkg788

Figure Lengend Snippet: Activity of FETUAcat constructs and TF abundance in human hepatoma cells. (A) The basal activity of p-3320/48cat and p-273/48cat in NCM as well as their responsiveness to CM was tested by transient transfection in HepG2 or Hep3B cells. Every Cat activity is relative to the activity (100%) of the pSV2cat plasmid (hatched bar) transfected in NCM-treated cells. For every cat construct, the CM-induced activation or repression relative to the reference activity in NCM-cultured cells is indicated by a broken arrow and a value (folds). (B) Amounts of NF-1 and C/EBP family members in nuclear extracts from Hep3B or HepG2 cells or rat liver. Total nuclear proteins (15 µg/lane) were separated by SDS–PAGE, electro-transferred and immunodetected with the antisera noted on the left. Each protein band is identified on the right with a p followed with the size (kDa) of an expected NF-1 or C/EBP isoform (35–37). A Coomassie blue staining of total proteins before electro-transfer is shown in the lower panel. At the bottom is the anode.

Article Snippet: The human HepG2 and Hep3B hepatoma cell lines (ATCC refs HB-8065 and HB-8064) were propagated as a monolayer in 75 cm 2 flasks containing DMEM/10% FCS supplemented as above, at 37°C under a 5% CO 2 -enriched and water-saturated atmosphere.

Techniques: Activity Assay, Construct, Transfection, Plasmid Preparation, Activation Assay, Cell Culture, SDS Page, Staining

Journal:

Article Title: The inflammation-induced down-regulation of plasma Fetuin-A (?2HS-Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighbouring binding sites

doi: 10.1093/nar/gkg788

Figure Lengend Snippet: Effect of exogenous C/EBP proteins upon the basal and CM-induced response of a FETUAcat plasmid. The FETUAcat plasmid p-273/48cat and either of various pC/EBP expression plasmids were co-transfected in the Hep3B cell line cultured in the presence of CM (dotted line) or NCM (solid line). The amounts of pC/EBPα, -β or -δ plasmids used for co-transfection are indicated on the abcissa. Every Cat activity is relative to the activity (100%) obtained in NCM-treated Hep3B cells that were transfected with an empty expression plasmid. The activities of pApoCIIIΔDcat and pGAPDHcat used as controls for a gene that is down-regulated by CM (APOCIII) or is not CM-responsive (GAPDH) are also shown.

Article Snippet: The human HepG2 and Hep3B hepatoma cell lines (ATCC refs HB-8065 and HB-8064) were propagated as a monolayer in 75 cm 2 flasks containing DMEM/10% FCS supplemented as above, at 37°C under a 5% CO 2 -enriched and water-saturated atmosphere.

Techniques: Plasmid Preparation, Expressing, Transfection, Cell Culture, Cotransfection, Activity Assay

Journal:

Article Title: The inflammation-induced down-regulation of plasma Fetuin-A (?2HS-Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighbouring binding sites

doi: 10.1093/nar/gkg788

Figure Lengend Snippet: Identification of a CM-responsive area in the FETUA promoter. (A) A series of cat plasmids with various deletions of the FETUA promoter were co-transfected with the expression plasmid pC/EBPα (100 ng) or an empty control plasmid pHD (100 ng) in Hep3B cells cultured with CM versus NCM. All values are relative to p-200/48cat activity (100%) in the presence of pHD and NCM. (B) p-200/48cat, pFETUAtkcat and pBLcat5 that harbour various segments of the FETUA and/or tk promoters as depicted, were co-transfected with pC/EBPα or pHD. The numbering –200, –43 and +48 refers to the FETUA gene. All values are relative to pBLcat5 activity (100%) in the presence of pHD and NCM.

Article Snippet: The human HepG2 and Hep3B hepatoma cell lines (ATCC refs HB-8065 and HB-8064) were propagated as a monolayer in 75 cm 2 flasks containing DMEM/10% FCS supplemented as above, at 37°C under a 5% CO 2 -enriched and water-saturated atmosphere.

Techniques: Transfection, Expressing, Plasmid Preparation, Cell Culture, Activity Assay

Journal: The American Journal of Pathology

Article Title: Lymphocyte-Specific Protein-1 Controls Sorafenib Sensitivity and Hepatocellular Proliferation through Extracellular Signal-Regulated Kinase 1/2 Activation

doi: 10.1016/j.ajpath.2018.06.005

Figure Lengend Snippet: Loss of LSP1 expression in the JM1 rat hepatoma cell line leads to increased sorafenib sensitivity with decreased cell numbers and phosphorylated extracellular signal-regulated kinase (pERK) expression. A: Representative bright-field images of scrambled and LSP1 shRNA JM1 hepatoma cells treated with dimethyl sulfoxide (DMSO), 20 μmol/L sorafenib, and 40 μmol/L sorafenib. B: Quantification of the concentration of DNA per well normalized to the DMSO control. C: Western blot analysis of pERK, total ERK, and proliferating cell nuclear antigen (PCNA) expression in scrambled and LSP1 shRNA JM1 cells. Ponceau S was used as a loading control. D: Quantification of the total number of cells per field of the LSP1 and scrambled control shRNA transfected Hep3B treated with 2 μmol/L sorafenib for 48 hours. E: Western blot analysis of total LSP1, PCNA, pERK, and total ERK in the LSP1 and scrambled shRNA transfected Hep3B treated with 2 μmol/L sorafenib or DMSO as control. ∗P < 0.05, ∗∗∗P < 0.001 versus scrambled. Original magnification, ×200 (A). −, DMSO control; +, sorafenib treated; C, untreated control (10, 20, and 40 μmol/L).

Article Snippet: Hep3B cells [Hep 3B2.1-7 (Hep 3B, Hep3-B, and Hep3B); HB-8064; ATCC, Manassas, VA] were transiently transfected with either human LSP1 shRNA or scrambled control shRNA (Origene; TG311654) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA).

Techniques: Expressing, shRNA, Concentration Assay, Western Blot, Transfection