Journal: ASPET Discovery

Article Title: Cheating Death: How a Porphyrin Transporter Fuels Survival in Hypoxia

doi: 10.1016/j.aspetd.2025.100011

Figure Lengend Snippet: Fig. 1. Hypoxia induces ABCB6 transcripts via a conserved HRE. (A) HepG2, NIH3T3, MEL, and K562 cells were cultured under normoxia or hypoxia (1% O2) for 48 hours. EPO and ABCB6 transcript levels were evaluated by RT-PCR. (B) Epo and Abcb6 transcript levels were determined by RT-PCR in livers and hearts of mice exposed to hypoxia (10% O2) or normoxia (20% O2) for 6 hours. (C) Changes in Abcb6 transcripts under 6 hours hypoxia was determined in mouse Hepa1c1c7 and BpRc1 (its somatic mutant derivative) cells that lack functional HIF1. (D) Abcb6 transcripts in MCF-7 cells exposed to hypoxia (16 hours) and treated with Hif-1α or Hif-2α siRNA obtained from GEO database (GSE3188) are shown. (E) WT mice or those in which HIF-1α was deleted in the liver were exposed to 6 hours of hypoxia (10% oxygen), and Abcb6 transcripts were analyzed in the liver by RT- PCR. (F) HIF1 transcriptional activity on Abcb6 promoter was tested with constructs lacking 1 of the 2 HREs (−136 or −238) as shown. Luciferase with Abcb6 promoter regions were cotransfected with HIF-1α, and luciferase activity was measured. (G) Chromatin immunoprecipitation of HIF-1α confirmed its binding to the -136 HRE under hypoxia. HRE, WT, wild type *P < .05, **P < .01, ***P < .001, ****P < .0001 (Student’s t test). UTR, untranslated region.

Article Snippet: Hepa-1c1c7, BpRcl, NIH/3T3, K562, mouse erythroleukemia (MEL), and HepG2 cells were obtained from the American Type Culture Collection.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Functional Assay, Activity Assay, Construct, Luciferase, Chromatin Immunoprecipitation, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B

doi: 10.1172/JCI141295

Figure Lengend Snippet: ( A ) Western blot for hASM and ( B ) quantification of hASM activity in lysates from HepG2 cells transduced with either control-AAV or hASM-AAV under a liver-specific promoter ( n = 3 independent replicates). ( C ) Quantification of hASM activity in culture supernatants from control-AAV– and hASM-AAV–infected HepG2 cells ( n = 3 independent replicates). ( D ) Confocal images of healthy and LGMD2B patient myoblasts prior to and following focal laser injury (site marked by white arrow) showing FM 1-43 dye (green) labeling. LGMD2B patient myoblasts were treated with culture supernatants from control and hASM-AAV–infected HepG2 cells in CIM. ( E ) Plot showing the averaged kinetics of FM-dye entry in healthy and patient myoblasts ( n > 15 cells per condition). Data are presented as mean ± SEM. * P < 0.001 (vs. control-AAV–treated cells) by independent samples t test ( B and C ) or mixed-model ANOVA with analyses for interaction effects between treatment condition and time was used ( E , vs. control-AAV–treated cell supernatant). Scale bars: 10 μm.

Article Snippet: HepG2 cell lysates were resolved in 4% to 12% gradient polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against ASM (rabbit polyclonal, Abcam, ab83354) and β-actin (mouse monoclonal, Santa Cruz Biotechnology, sc47778).

Techniques: Western Blot, Activity Assay, Transduction, Control, Infection, Labeling

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