Transfection of adult primary rat hepatocytes in culture

Gardmo, Cissi; Kotokorpi, Pia; Helander, Hanna; Mode, Agneta

doi:10.1016/j.bcp.2005.03.028

Cited by 22 publications

(21 citation statements)

References 22 publications

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“…Passages lower than 10 were used. Cells stably transfected with FGFR-2-CA (2 independent bulk transfections) or with the empty vector (1 Â 10 6 cells) (19) were inoculated orthotopically along with matrigel, into the mammary gland 4 of NOD/SCID mice (n ¼ 2; 4 animals per group).…”

Section: Cell Linesmentioning

confidence: 99%

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

et al. 2011

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Fibroblast growth factor (FGF) receptor 2 (FGFR-2) polymorphisms have been associated with an increase in estrogen receptor and progesterone receptor (PR)-positive breast cancer risk; however, a clear mechanistic association between FGFR-2 and steroid hormone receptors remains elusive. In previous works, we have shown a cross talk between FGF2 and progestins in mouse mammary carcinomas. To investigate the mechanisms underlying these interactions and to validate our findings in a human setting, we have used T47D human breast cancer cells and human cancer tissue samples. We showed that medroxyprogesterone acetate (MPA) and FGF2 induced cell proliferation and activation of ERK, AKT, and STAT5 in T47D and in murine C4-HI cells. Nuclear interaction between PR, FGFR-2, and STAT5 after MPA and FGF2 treatment was also showed by confocal microscopy and immunoprecipitation. This effect was associated with increased transcription of PRE and/or GAS reporter genes, and of PR/STAT5-regulated genes and proteins. Two antiprogestins and the FGFR inhibitor PD173074, specifically blocked the effects induced by FGF2 or MPA respectively. The presence of PR/FGFR-2/ STAT5 complexes bound to the PRE probe was corroborated by using NoShift transcription and chromatin immunoprecipitation of the MYC promoter. Additionally, we showed that T47D cells stably transfected with constitutively active FGFR-2 gave rise to invasive carcinomas when transplanted into NOD/SCID mice. Nuclear colocalization between PR and FGFR-2/STAT5 was also observed in human breast cancer tissues. This study represents the first demonstration of a nuclear interaction between FGFR-2 and STAT5, as PR coactivators at the DNA progesterone responsive elements, suggesting that FGFRs are valid therapeutic targets for human breast cancer treatment. Cancer Res; 71(10); 3720-31. Ó2011 AACR.

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Section: Cell Linesmentioning

confidence: 99%

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

et al. 2011

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“…Small interfering ribonucleic acid transfection was carried out as described by Gardmo et al (2005). Cells were grown in each dish until they reached 75% confluence.…”

Section: Small Interfering Ribonucleic Acid Transfectionmentioning

confidence: 99%

Interleukin‐6 promotes 2‐deoxyglucose uptake through p44/42 MAPKs activation via Ca²⁺/PKC and EGF receptor in primary cultured chicken hepatocytes

Lee

Han

2008

Journal Cellular Physiology

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Interleukin-6 (IL-6) is involved in a variety of biological responses, including the glucose metabolism and cell growth, which is a critical physiological function requiring multiple metabolic pathways. Therefore, in the present study, we examined the effect of IL-6 on 2-deoxyglucose (2-DG) uptake and the related signaling pathways in primary cultured chicken hepatocytes. IL-6 increased 2-DG uptake in a time- (> or =4 h) and a dose -(> or =5 ng/ml) dependent manner. Indeed, IL-6 increased GLUT-2 mRNA and protein expression as well as 2-DG uptake, which were blocked by actinomycin D (AD, transcription inhibitor) and cycloheximide (CHX, translation inhibitor). IL-6 (10 ng/ml) increased the level of IL-6Ralpha and glycoprotein (gp) 130 (IL-6Rbeta) protein expressions. IL-6 increased Janus Kinase (JAK)-2, signal transducer and activator of transcription (STAT)-3 phosphorylation, intracellular Ca(2+) concentration, and PKC phosphorylation. IL-6-induced increase of 2-DG uptake and GLUT-2 protein expression were blocked by JAK2-specific siRNA, a STAT3 inhibitor, staurosporine, and bisindolylmaleimide I (PKC inhibitors). In addition, IL-6 increased EGFR/src/FAK, PI3K/Akt phosphorylation and 2-DG uptake as well as GLUT-2 protein expression, which were blocked by AG 1478 (EGF receptor inhibitor), PP2 (src family of tyrosine kinase inhibitor), PI3K-specific siRNA, and a Akt inhibitor. Furthermore, IL-6 increased p44/42 MAPKs phosphorylation and p44 and p42 MAPK-specific siRNA mixture blocked IL-6-induced increase of 2-DG uptake and GLUT-2 protein expression. In conclusion, IL-6 stimulates the 2-DG uptake through p44/42 MAPKs activation via Ca(2+)/PKC and EGF receptor in primary cultured chicken hepatocytes.

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“…Unfortunately, primary human hepatocytes are not amenable to siRNA transfection or protein labeling [16], [17], [18]. Therefore to investigate the effects of JNK and p38 inhibition upon CD154∶ROS mediated human hepatocyte apoptosis we used specific JNK and p38 inhibitors [19], [20], [21].…”

Section: Resultsmentioning

confidence: 99%

Activation of CD40 with Platelet Derived CD154 Promotes Reactive Oxygen Species Dependent Death of Human Hepatocytes during Hypoxia and Reoxygenation

et al. 2012

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BackgroundHypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.MethodsHuman hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.ResultsExposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.ConclusionsCD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.

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Transfection of adult primary rat hepatocytes in culture

Cited by 22 publications

References 22 publications

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

Interleukin‐6 promotes 2‐deoxyglucose uptake through p44/42 MAPKs activation via Ca²⁺/PKC and EGF receptor in primary cultured chicken hepatocytes

Activation of CD40 with Platelet Derived CD154 Promotes Reactive Oxygen Species Dependent Death of Human Hepatocytes during Hypoxia and Reoxygenation

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Transfection of adult primary rat hepatocytes in culture

Cited by 22 publications

References 22 publications

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

Interleukin‐6 promotes 2‐deoxyglucose uptake through p44/42 MAPKs activation via Ca2+/PKC and EGF receptor in primary cultured chicken hepatocytes

Activation of CD40 with Platelet Derived CD154 Promotes Reactive Oxygen Species Dependent Death of Human Hepatocytes during Hypoxia and Reoxygenation

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Interleukin‐6 promotes 2‐deoxyglucose uptake through p44/42 MAPKs activation via Ca²⁺/PKC and EGF receptor in primary cultured chicken hepatocytes