cell line culture aml12 cell  (ATCC)

Journal: Cancer research

Article Title: Obesity/type 2 diabetes-associated liver tumors are sensitive to Cyclin D1 deficiency

doi: 10.1158/0008-5472.CAN-20-0106

Figure Lengend Snippet: A. Metformin prevents insulin-induced Cyclin D1 protein levels in hepatocytes. Overnight serum starved AML12 murine hepatocytes were treated with control, metformin (10 mM), insulin (200 nM) or both for 4 h, followed by immunoblotting to detect Cyclin D1 expression. B. Design and metabolic phenotypes of the metformin treatment experiment. Metformin was given at 50 mg/kg for 27 weeks. Endpoint blood insulin level and liver histology of the db/db mice were shown. Scale bar represents 500 μm. C. Metformin treatment prevents the induction of Cyclin D1 in type 2 diabetic liver as detected by immunoblotting. D. Metformin treatment protects mice from DEN-induced liver cancer in diabetic mice (water, n=5; metformin, n=4).

Article Snippet: Tissue Culture AML12 murine hepatocyte cell line was obtained from ATCC, and maintained in DMEM/F12 (Sigma-Aldrich) with 10% FBS, 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone, 100 U/ml penicillin, and 100 mg/ml streptomycin.

Techniques: Control, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

doi: 10.1074/jbc.M116.744631

Figure Lengend Snippet: Inhibition of Habp2 mRNA expression by TGF-β. A, murine AML12 hepatocyte cell line (black bars) and murine primary hepatocytes (gray bars) were stimulated with epidermal growth factor (EGF, 50 ng/ml), platelet-derived growth factor (PDGF-BB, 50 ng/ml), basic fibroblast growth factor (bFGF, 10 ng/ml), hepatocyte growth factor (HGF, 10 ng/ml), connective tissue growth factor (CTGF, 50 ng/ml), transforming growth factor-β (TGF-β, 100 ng/ml), interleukin-1β (IL-1β, 20 ng/ml), estrogen (Estr., 10 μg/ml), and progesterone (Prog., 10 μg/ml) for 24 h. Relative Habp2 mRNA levels were normalized to Gapdh and represent mean ± S.D. (n = 3). B and C, AML12 cells (square) and primary hepatocytes (circles) were treated with 100 ng/ml of TGF-β for increasing time (B), or with increasing concentrations of TGF-β for 24 h (C) and Habp2 mRNA levels were quantified. Values are mean ± S.E. (n = 3) of a single experiment and similar results were obtained in three independent experiments. Statistically significant results (p < 0.05) compared with untreated cells as measured by ANOVA is denoted by *.

Article Snippet: Cell Culture Mouse hepatocyte cell line AML12 (ATCC® CRL-2254 TM ) was cultivated in DMEM (Invitrogen, Darmstadt, Germany) with 10% ( v / v ) fetal calf serum (FCS), Thermo-Fisher Scientific (Fermentas), Rockford), 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen) on cell culture-treated plastic (Nunc, Wiesbaden, Germany).

Techniques: Inhibition, Expressing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

doi: 10.1074/jbc.M116.744631

Figure Lengend Snippet: Inhibition of TGF-β Type-I receptor (ALK5), but not SMAD3 blocks the effects of TGF-β. AML12 cell line (A) and primary hepatocytes (B) were pre-treated with SB 431542 (10 μm), SIS3 (10 μm) or DMSO for 30 min before stimulation with TGF-β (100 ng/ml) for 24h (white bars) or were left unstimulated (black bars). qPCR analysis of Habp2 mRNA expression was performed and represent mean ± S.E. (n = 3). Statistically significant results (p < 0.05) compared with untreated cells as measured by ANOVA are denoted by *. C, Western blotting analysis of FSAP in the supernatant of AML12 cells as well as phospho SMAD3 and SMAD2/3 in the cell extracts. D, primary hepatocytes were pre-treated with SB 431542 (10 μm) for 30 min before stimulated with TGF-β (100 ng/ml) for 24 h. Cells were fixed with paraformaldehyde and stained against FSAP (green) and nuclei (DAPI, blue). Similar results were obtained in two independent experiments.

Article Snippet: Cell Culture Mouse hepatocyte cell line AML12 (ATCC® CRL-2254 TM ) was cultivated in DMEM (Invitrogen, Darmstadt, Germany) with 10% ( v / v ) fetal calf serum (FCS), Thermo-Fisher Scientific (Fermentas), Rockford), 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen) on cell culture-treated plastic (Nunc, Wiesbaden, Germany).

Techniques: Inhibition, Expressing, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

doi: 10.1074/jbc.M116.744631

Figure Lengend Snippet: Effect of inhibition of signaling pathways on the expression of Habp2 by TGFβ. A, AML-12 cells were transfected with control siRNA or Smad2 and Smad3 for 24 h. Cells were then stimulated with TGF-β (100 ng/ml) for 24 h before analysis of Habp2 mRNA. Expression relative to Gapdh is shown, and values are mean ± S.D. (n = 3) of a single experiment and similar results were obtained in two independent experiments. B, AML12 cell were pre-treated with Ly294002 (10 μm), PD98059 (10 μm), SB203580 (10 μm), and DMSO for 30 min before stimulation with TGF-β (100 ng/ml) for 24 h or were left unstimulated. qPCR analysis of Habp2 mRNA expression was performed and represent mean ± S.E. (n = 3). Statistically significant results (p < 0.05) compared with untreated cells as measured by ANOVA is denoted by *. Similar results were obtained in two independent experiments.

Article Snippet: Cell Culture Mouse hepatocyte cell line AML12 (ATCC® CRL-2254 TM ) was cultivated in DMEM (Invitrogen, Darmstadt, Germany) with 10% ( v / v ) fetal calf serum (FCS), Thermo-Fisher Scientific (Fermentas), Rockford), 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen) on cell culture-treated plastic (Nunc, Wiesbaden, Germany).

Techniques: Inhibition, Protein-Protein interactions, Expressing, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

doi: 10.1074/jbc.M116.744631

Figure Lengend Snippet: Activity of human HABP2 promoter reporter constructs. A, indicates details of the HABP2 promoter fragments used. B, basal promoter activity of the human HABP2 promoter fragments of various lengths was tested in AML12 hepatocytes. Significant changes relative to the −1/−500 fragment are indicated by *. C, cells were pre-treated with SB 431542 (10 μm) or 30 min before stimulated with TGF-β (100 ng/ml) for 24h before lysis. Activity is shown as mean ± S.E. (n = 5). Since the basal expression of the different constructs varied, the activities of the treated samples are normalized to the untreated sample for that particular construct. Statistically significant difference (p < 0.05) between control and TGF-β as measured by ANOVA is denoted by *. Similar results were obtained in three independent experiments.

Article Snippet: Cell Culture Mouse hepatocyte cell line AML12 (ATCC® CRL-2254 TM ) was cultivated in DMEM (Invitrogen, Darmstadt, Germany) with 10% ( v / v ) fetal calf serum (FCS), Thermo-Fisher Scientific (Fermentas), Rockford), 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen) on cell culture-treated plastic (Nunc, Wiesbaden, Germany).

Techniques: Activity Assay, Construct, Lysis, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

doi: 10.1074/jbc.M116.744631

Figure Lengend Snippet: c-fos interacts with the minimal Habp2 promoter. A, DNA sequence representing the minimally active and TGF-β-responsive promoter region (−1/−177) and other non-responsive regions (−313/−427 and −177/−313) were biotinylated and incubated with nuclear extracts from AML12 cells. After pull-down with streptavidin magnetic beads the bound fraction was subjected to Western blotting analysis with an anti-c-fos antibody or anti-c-jun antibody. Band density was quantified, and results represent mean ± S.E. (n = 3). B/C, ChIP was performed on nuclear extracts of AML12 cells with or without TGF-β stimulation for 24 h. Pull down was performed with an anti-c-fos (B) or an anti-SMAD 2/3 (C) antibody. Intact extract was used as an input control. PCR was performed to amplify the minimally active and TGF-β-responsive promoter region (−1/−177) and other non-responsive region (−313/−427). Values represent mean ± S.E. (n = 3 independent experiments). Statistically significant results (p < 0.05) compared with untreated cells as measured by ANOVA is denoted by *.

Article Snippet: Cell Culture Mouse hepatocyte cell line AML12 (ATCC® CRL-2254 TM ) was cultivated in DMEM (Invitrogen, Darmstadt, Germany) with 10% ( v / v ) fetal calf serum (FCS), Thermo-Fisher Scientific (Fermentas), Rockford), 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen) on cell culture-treated plastic (Nunc, Wiesbaden, Germany).

Techniques: Sequencing, Incubation, Magnetic Beads, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

doi: 10.1074/jbc.M116.744631

Figure Lengend Snippet: Deletion mutations within the 177-bp HABP2 promoter. A, transcription factor binding sites were deleted from the minimally active and TGF-β-responsive promoter region (−1/−177) as indicated in Fig. 6A. B, basal reporter activity was measured in AML12 cells. C, reporter activity was measured in the absence or presence of TGF-β (10 ng/ml) for 24 h. Values represent mean ± S.E. (n = 3). Since the basal expression of the different constructs varied, the activities of the treated samples are normalized to the untreated sample for that particular construct. Statistically significant results (p < 0.05) compared with TGF-β-treated cells transfected with the control (−1/−177) construct was determined by ANOVA and is denoted by *.

Article Snippet: Cell Culture Mouse hepatocyte cell line AML12 (ATCC® CRL-2254 TM ) was cultivated in DMEM (Invitrogen, Darmstadt, Germany) with 10% ( v / v ) fetal calf serum (FCS), Thermo-Fisher Scientific (Fermentas), Rockford), 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen) on cell culture-treated plastic (Nunc, Wiesbaden, Germany).

Techniques: Binding Assay, Activity Assay, Expressing, Construct, Transfection, Control