Journal:

Article Title: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

doi: 10.1101/gad.1529107

Figure Lengend Snippet: Induction of glycolysis by hypoxia is dependent on growth factor signals. (A) Cells were cultured in the presence or absence (3 wk) of IL-3. Following 24 h incubation at 21% O2 or 1.5% O2, cellular glucose utilization was measured by the conversion of 5-3H-glucose to 3H2O. Data represent mean of three independent experiments ± SD. (B) Glucose-dependent lipid synthesis was measured by labeling cells with D-[U-14C6] glucose for 24 h. Lipid extracts were obtained from 2 × 106 cells as described in the Materials and Methods. Data are a representative experiment performed in triplicate ± SD.

Article Snippet: For experiments performed in the presence of IL-3, complete medium was supplemented with 3.5 ng/mL recombinant mouse IL-3 (BD Pharmingen).

Techniques: Cell Culture, Incubation, Labeling

Journal:

Article Title: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

doi: 10.1101/gad.1529107

Figure Lengend Snippet: Growth factors regulate the expression and activity of HIF-1α. (A) Cells cultured in the presence (+IL3) or absence of IL-3 at the indicated time points were incubated in normoxic (N) or hypoxic (H) conditions for 4 h and HIF-1α protein levels were analyzed by Western blot. Cells cultured in the presence of IL-3 and treated with 100 μM CoCl2 were used as a control. (B) At similar time points following withdrawal from IL-3, cells were treated with PBS or 100 μM CoCl2 for 4 h and analyzed by Western blot for HIF-1α and PDK-1 protein levels. Lysates from cells cultured in the presence of IL-3 exposed to hypoxia were used as a control. STAT3 was used as a loading control. Cells were cultured in the presence or absence of IL-3, and at the indicated time points, total RNA was isolated and analyzed for Hif-1α (C) or Glut1 (D) mRNA levels by real-time qPCR. Data are a representative experiment for triplicate samples ± SD. (E) HIF-1α promoter activity in response to hypoxia is lost during growth factor withdrawal. HIF-1α-responsive promoter containing three tandem HRE from the murine pgk1 was transfected into cells cultured in the presence or absence (3 wk) of IL-3. Ten hours after transfection, cells were incubated in normoxic (21% O2) or hypoxic (1.5% O2) conditions for an additional 24 h. Promoter activity is expressed as a ratio of luciferase to Renilla activity (relative luminescence units). Data are a representative experiment ± SD. for triplicate samples.

Article Snippet: For experiments performed in the presence of IL-3, complete medium was supplemented with 3.5 ng/mL recombinant mouse IL-3 (BD Pharmingen).

Techniques: Expressing, Activity Assay, Cell Culture, Incubation, Western Blot, Isolation, Transfection, Luciferase

Journal:

Article Title: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

doi: 10.1101/gad.1529107

Figure Lengend Snippet: Control of glycolytic recovery following growth factor restimulation does not involve HIF-1α expression. (A) Glycolytic rate of cells cultured in the absence of growth factor for 4 wk was measured at the indicated time points following readdition of IL-3. Graph is a representative experiment plotted as the mean ± SD of one triplicate experiment. (B) At various time points following IL-3 readdition, cells were subjected to normoxic or hypoxic conditions for an additional 4 h. Cell lysates were collected and Western blot analysis was used to measure HIF-1α and PDK-1 protein levels. STAT3 was used as a loading control.

Article Snippet: For experiments performed in the presence of IL-3, complete medium was supplemented with 3.5 ng/mL recombinant mouse IL-3 (BD Pharmingen).

Techniques: Expressing, Cell Culture, Western Blot

Journal:

Article Title: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

doi: 10.1101/gad.1529107

Figure Lengend Snippet: Cells expressing stable shRNA targeting HIF-1α have impaired survival but enhanced proliferative capacity. (A) Real-time qPCR analysis of Hif-1α mRNA levels. RNA from cells was isolated and cDNA synthesis was performed as described in the Materials and Methods section. Data are a representative experiment. (B) Western blotting of HIF-1α and PDK-1 in clones stably expressing shRNA to HIF-1α after culturing for 4 h in normoxic (N) or hypoxic (H) conditions. Lysates from vector cells cultured in the presence of IL-3 and exposed to 100 μM CoCl2 were used as a positive control. (C) Viability of HIF-1α knockdown cells grown in the presence of IL-3 cultured under hypoxic condition. At each time point, cells were collected and viable cells were assessed by staining with propidium iodide. Data represent mean of three independent experiments ± SD. (D) Viability of HIF-1α knockdown cells grown in the absence of IL-3 cultured under hypoxic condition. At each time point, cells were collected and viable cells assessed by staining with propidium iodide. Data represent mean of three independent experiments ± SD. (E) Growth curves of IL-3-stimulated cells expressing stable HIF-1α knockdown cultured under normoxic conditions. Data are a representative experiment performed in triplicate ± SD. (F) The rate of oxygen consumption under normoxia was measured in HIF-1α knockdown cells cultured in the presence of IL-3. Data are a representative experiment performed in triplicate ± SD. (G) Mitochondrial membrane potential was measured in cells cultured in the presence of IL-3 using the potentiometric dye TMRE. The value in the top right corner is a representative value of the mean channel fluorescence (MCF). Unstained cells and stained cells are represented by light and bold histograms, respectively.

Article Snippet: For experiments performed in the presence of IL-3, complete medium was supplemented with 3.5 ng/mL recombinant mouse IL-3 (BD Pharmingen).

Techniques: Expressing, shRNA, Isolation, Western Blot, Clone Assay, Stable Transfection, Plasmid Preparation, Cell Culture, Positive Control, Staining, Fluorescence

Journal:

Article Title: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

doi: 10.1101/gad.1529107

Figure Lengend Snippet: Cell growth and proliferation are enhanced in the absence of HIF-1α. Cells were withdrawn from IL-3 and size (A) and cell number (B) were measured at the indicated time points. On day 16 of withdrawal, cells were restimulated with IL-3 and size (C) and cell number (D) were measured. Data represent the mean of three independent experiments ± SD. (E) Cells cultured in the absence of IL-3 for 16 d were placed in fresh complete medium containing IL-3. At the various time points, cells were subjected to an additional 4 h of culture under 21% O2 (N) or 1.5% O2 (H). Cell lysates were analyzed by Western blot for HIF-1α and PDK-1 expression. A representative vector control and a HIF-1α cell line (C18) expressing a stable HIF-1α shRNA are shown. STAT3 was used as a loading control. Cells cultured in the presence of IL-3 and exposed to hypoxia were used as a positive control.

Article Snippet: For experiments performed in the presence of IL-3, complete medium was supplemented with 3.5 ng/mL recombinant mouse IL-3 (BD Pharmingen).

Techniques: Cell Culture, Western Blot, Expressing, Plasmid Preparation, shRNA, Positive Control

Journal:

Article Title: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

doi: 10.1101/gad.1529107

Figure Lengend Snippet: Induction of glycolysis through HIF-1α is anti-proliferative. (A) Growth factors stimulate cellular glucose uptake and the expression of HIF-1α. Under conditions of normoxia, the accumulation of HIF-1α is repressed by O2-dependent hydroxylation and degradation of the HIF-1α protein. When HIF-1α is repressed by O2-dependent degradation and/or HIF-1α shRNA, glycolytic pyruvate is diverted into mitochondrial-dependent lipid synthesis. Pyruvate is catabolized by mitochondrial PDH into acetyl-CoA, which is used in the TCA cycle to produce citrate that is transported into the cytosol, where it acts as a further regulator of glucose-6-P metabolism. Citrate is metabolized to produce acetyl-CoA by the enzyme ATP-citrate lyase (ACL) to supply the cell with a source of acetyl-CoA for lipid synthesis. When HIF-1α is stabilized by mitochondrial ROS or oxygen deprivation, the increased HIF-1α-dependent transcription can result in a metabolic reprogramming of intracellular glucose fate. The HIF-1α-dependent increases in glycolytic genes including Ldh-A lead to an enhanced rate of anaerobic glycolysis, and the induction of mitochondrial regulatory enzymes such as PDK-1 decrease pyruvate metabolism in the mitochondria, resulting in decreased mitochondrial TCA cycle activity and cytosolic citrate levels. These effects would support non-oxygen-dependent ATP production by degradation of glucose to lactate, and suppress mitochondrial activity and cytosolic lipid synthesis. (B) Glycolytic rate of cells cultured in the presence of IL-3 following 24 h incubation under 21% O2 or 1.5% O2. In the absence of IL-3 (not shown), glycolysis was reduced <6.58 nmol of glucose per hour in both the vector controls and HIF-1α shRNA cells. Data are the mean of three independent experiments ± SD performed in triplicate. (C) Cells cultured in the presence of IL-3 were subjected to 4 h of 21% O2 or 1.5% O2 followed by an additional 20–24 h incubation with 14C-labeled pyruvate. Lipids were extracted from cell lysates and total incorporation of 14C-labeled lipid was measured by scintillation counting. Data are a representative experiment ± SD for triplicate samples. (D) Accumulation of lactate in cell culture supernatants. Cells were cultured in 1 mL of medium for 5 d in the presence of IL-3 and subjected to 1.5% O2 for an additional 24 h. Lactate levels in supernatants were measured as described in Materials and Methods. Data represent three independent experiments ± SD.

Article Snippet: For experiments performed in the presence of IL-3, complete medium was supplemented with 3.5 ng/mL recombinant mouse IL-3 (BD Pharmingen).

Techniques: Expressing, shRNA, Activity Assay, Cell Culture, Incubation, Plasmid Preparation, Labeling

Journal: Cell stem cell

Article Title: M 6 A demethylase ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in acute myeloid leukemia

doi: 10.1016/j.stem.2020.04.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant mouse IL-3 , PeproTech , Cat#213–13.

Techniques: Recombinant, Control, Virus, Magnetic Beads, Reverse Transcription, Proliferation Assay, Purification, Real-time Polymerase Chain Reaction, Software