Journal: Endocrinology

Article Title: Endogenous IGFBP-3 Mediates Intrinsic Apoptosis Through Modulation of Nur77 Phosphorylation and Nuclear Export.

doi: 10.1210/en.2015-1215

Figure Lengend Snippet: Figure 1. Endogenous Nur77 is knocked down using two different siRNA oligos and is detectable in MAC-T cells with a commercially available antibody. Cells were transfected with scramble (SCR) or Nur77 siRNA (Oligo No. 1 or Oligo No. 2). Total RNA (A) or protein (B) was isolated from WCLs after 48 h and analyzed for Nur77 by RT-qPCR (corrected for cyclophilin) or Western immunoblot (50 g protein), respectively. For the immunoblot, HSP60 served as a loading control. Bars represent mean SE of three experiments. C, To confirm specificity of the Nur77 antibody, MAC-T WCL (50 g), WCL from MAC-T cells transfected with myc-tagged Nur77 (3 g), HepG2 WCL (50 g), and nuclear fractions from MAC-T cells (5 g) were immunoblotted for Nur77 on one half of the membrane and myc on the other half. Actin served as a loading control.

Article Snippet: HepG2 cell lysate was obtained from Novus Biologicals.

Techniques: Transfection, Isolation, Quantitative RT-PCR, Western Blot, Control, Membrane

Journal: Methods in Molecular Biology

Article Title: Transcription Factors

doi: 10.1007/978-1-60761-738-9

Figure Lengend Snippet: Fig. 1. Inhibition of HIF-hydroxylase activity by CoCl2. (a) In vitro prolyl hydroxylase activity assay. The GST-HIF1a-TADN fusion protein or the GST protein was incubated with HepG2 cell extract, cofactors, and [5-14C]2-oxoglutarate in the presence of CoCl2 (10 mM). The radioactivity associated to 14C-succinate was determined. In each experi- ment, the basal HIF-TADN-dependent activity (control) was set to 100% after being normalized by subtracting the GST-associated activity. Values are means ± SEM of three independent culture experiments. Statistics, Student’s t-test for paired values: *P £ 0.05 vs. control. (b) GST pull-down assay. HepG2 cells were treated with or without CoCl2 (10 mM). Cell extracts were prepared and incubated with the GST-HIF1a-TADN fusion protein supplemented with cofactors. Glutathione-Sepharose beads and [35S]VHL were then added and the bound VHL was recovered, subjected to SDS–PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]VHL. The two bands represent the 213 and 160 amino acid VHL translation products (105, 106).

Article Snippet: 100 μg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1.

Techniques: Inhibition, Activity Assay, In Vitro, Incubation, Radioactivity, Control, Pull Down Assay, SDS Page

Journal: Methods in Molecular Biology

Article Title: Transcription Factors

doi: 10.1007/978-1-60761-738-9

Figure Lengend Snippet: Fig. 2. Inhibition of the IGF-1-mediated HIF-1alpha induction by the PI(3)-kinase inhibitor LY294002 and the MEK inhibitor U0126. Serum-starved HepG2 cells were pretreated with 10 µM LY294002 or 10 µM U0126 for 30 min and then treated either with or without 100 nM human IGF-1 (Sigma) and exposed to normoxia (16% O2) or hypoxia (8% O2) for 4 h. Acetic acid was used in controls at a final concentration of 100 nM to keep the pH constant. 100 µg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1. Autoradiographic signals were detected by chemiluminescence (77).

Article Snippet: 100 μg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1.

Techniques: Inhibition, Concentration Assay, Western Blot, Transduction

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on hepatic apoC-III expression. Rat primary hepatocytes were transduced with Foxo1 or LacZ vector at an MOI of 50 PFU/cell or mock-transduced with PBS. After 24 hours of transduction, the intracellular levels of apoC-III (A), Foxo1 (B), and GK (C) mRNA were determined by real-time RT-PCR using β-actin mRNA as control. The effect of Foxo1 on hepatic apoC-III expression in response to insulin was assayed in HepG2 cells. Cells were transduced with Foxo1, Foxo1-ADA, or control LacZ vector (50 PFU/cell) in the absence or presence of insulin at different concentrations. Twenty-four hours after transduction, cells were collected for determination of the intracellular levels of apoC-III mRNA induced by Foxo1 (D) and Foxo1-ADA (E). *P < 0.05, **P < 0.005; significantly different from controls. NS, not significant by ANOVA. Data were from 3 independent experiments.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Expressing, Transduction, Plasmid Preparation, Quantitative RT-PCR

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on the human APOC3 promoter activity. (A) The APOC-III promoter–directed luciferase reporter system. The wild-type and mutant IRE sequences are underlined. (B) Foxo1-mediated induction of the APOC3 promoter activity. HepG2 cells were transfected by pHD317 together with Foxo1 construct, or with both Foxo1 and Foxo1-Ø256 constructs. For each construct, 1 μg of DNA for each construct was used in transfection. For normalization of transfection efficiency, 1 μg pCMV5-LacZ DNA was included for normalization of transfection efficiency. (C) The APOC3 promoter variants in the luciferase reporter system. (D) Responses of APOC3 promoter variants to Foxo1 production. HepG2 cells were transfected with individual test plasmids in the absence (–) or presence (+) of pCMV5-Foxo1. The relative luciferase activity, after normalizing to β-gal activity, was compared between basal (–) and Foxo1-inducible (+) conditions. (E) Responses of wild-type and mutant APOC3 promoters to insulin. Test plasmids were transduced into HepG2 cells in the presence and absence of pCMV5-Foxo1 transfection in culture media, either supplemented with or without insulin (30 nM). The relative luciferase activity in transduced cells was determined using β-gal activity as control. *P < 0.001 vs. controls.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Activity Assay, Luciferase, Mutagenesis, Transfection, Construct

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Molecular interaction between Foxo1 and the APOC3 promoter. Molecular association between Foxo1 and the APOC3 promoter was analyzed by EMSA and ChIP. Aliquots of Foxo1 protein from linked in vitro transcription-translation products (5 μg) were incubated with 2.5 μl of radioactively labeled DNA corresponding to –467/–440 nt in the human APOC3 promoter (WT-IRE) (A), a mutant APOC3 IRE (mt-IRE) containing 2 substitutions, of –A458C and –A460G, and a control PEPCK IRE DNA (B), followed by electrophoresis through 8% nondenaturing polyacrylamide gels for 30 minutes. Lane 1, DNA probe alone. Lane 2, DNA probe + Foxo1 protein lysates. Lane 3, DNA probe + Foxo1 protein lysates + anti-Foxo1 antibody (1 μg). Lane 4, DNA probe + Foxo1 protein lysates + nonlabeled competitor DNA at a molar concentration of 50-fold excess. Free, shifted, and supershifted DNA bands were visualized by autoradiography. For ChIP assay, HepG2 cells were transduced with Foxo1 vector at an MOI of 50 PFU/cell. Cells were harvested 24 hours later and subjected to ChIP using PBS as a negative control (lane 5), control IgG (lane 6), and anti-Foxo1 antibody (lane 7). The coimmunoprecipitated chromatin DNA was analyzed by immunoblot (C) using anti-Foxo1 antibody and PCR (D) using the primers that correspond to –655/–20 nt of the APOC3 promoter.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: In Vitro, Incubation, Labeling, Mutagenesis, Electrophoresis, Concentration Assay, Autoradiography, Transduction, Plasmid Preparation, Negative Control, Western Blot

Journal: PLoS ONE

Article Title: Glucose-coated superparamagnetic iron oxide nanoparticles prepared by metal vapor synthesis can target GLUT1 overexpressing tumors: In vitro tests and in vivo preliminary assessment

doi: 10.1371/journal.pone.0269603

Figure Lengend Snippet: GLUT1 levels estimated by ELISA in PSN-1, BCPAP and HEK293 cells (at the top). Uptake studies: PSN-1 and BCPAP cells were incubated with 0.1 mg/mL of Glc-SPIONs for 30 min, 1, 3, 6, and 12 h. The Fe cellular content was estimated by GF-AAS analysis. Uptake studies after treating the cells with GLUT1 inhibitors: PSN1 and BCPAP cells were pre-incubated for 1 h with anti-GLUT1 polyclonal antibody, WZB117, Fasentin, BAY-876, and STF-31. Subsequently, cells were treated for 3 or 6 h with 0.1 mg/mL of Glc-SPIONs.

Article Snippet: Expression levels in cancer cells were evaluated by means of the GLUT1 Colorimetric Cell-Based ELISA kit (Boster Biological Technology, Pleasanton CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: Antioxidants

Article Title: NRF2 Activation Ameliorates Oxidative Stress and Improves Mitochondrial Function and Synaptic Plasticity, and in A53T α-Synuclein Hippocampal Neurons

doi: 10.3390/antiox11010026

Figure Lengend Snippet: DMF activates NRF2 in vitro and this activation is inhibited by ML385. ( A ) 20 µM DMF significantly induces NRF2 expression in HepG2-ARE cells, and co-treatment with 1 µM ML385 significantly inhibits this effect. ( B ) 20 µM DMF significantly induced expression of NRF2-ARE genes, GCLC, HMOX1, and NQO1 in WT and A53TSyn neurons ( n = 10–12). Significance is relative to control unless otherwise indicated ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: HepG2 cells that stably express a firefly luciferase gene under the control of the ARE promoter were obtained from BPS Bioscience.

Techniques: In Vitro, Activation Assay, Expressing

Journal: The EMBO Journal

Article Title: SLC25A1 and ACLY maintain cytosolic acetyl-CoA and regulate ferroptosis susceptibility via FSP1 acetylation

doi: 10.1038/s44318-025-00369-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: A375, SK-MEL-28, SK-MEL-30, HT29, RKO, Huh-7, HepG2 and A549 cells were infected with lentiviral for 24 h, followed by selecting with fresh medium containing 2 μg/mL puromycin (InvivoGen, ant-pr-1) to obtain stably infected cells.

Techniques: Membrane, Magnetic Beads, Recombinant, Control, Sequencing, Software, Cell Viability Assay, Staining, Purification, SYBR Green Assay, Citrate Assay, Isolation, GSSG Assay, AST Assay, Enzyme-linked Immunosorbent Assay