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1.
Figure 3.

Figure 3. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

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.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
2.
Figure 1.

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

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.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
3.
Figure 6.

Figure 6. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

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 Cells were withdrawn from IL-3 and size (A) and cell number (B) were measured at the indicated time points.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.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
4.
Figure 8.

Figure 8. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

Glucose utilization in primary T cells is impaired by HIF-1α. (A) Glucose consumption in primary T cells isolated from Hif-1αfl/fl Cre (wild-type [WT]) or Hif-1fl/fl Cre+ (Cre) mice that were administered tamoxifen. T cells were stimulated under 21% or 1.5% oxygen by plate-bound anti-CD3/anti-CD28 in 1-mL cell cultures, and supernatants were collected 48 h post-stimulation for glucose analysis. Data are a representative experiment performed in triplicate ± SD. (B) Cells were activated by plate-bound anti-CD3/ anti-CD28 for 2 d followed by addition of 14C-labeled pyruvate under normoxic or hypoxic conditions. Cells were cultured for an additional 2 d in the presence of IL-2. 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. (C) Cells were cultured under identical conditions described in A and the amount of secreted lactate into 1 mL of culture medium was measured as described in Materials and Methods. Data are a representative experiment ± SD.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
5.
Figure 5.

Figure 5. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

Cellular proliferation is controlled by the levels of HIF-1α. (A) Growth curves of primary T cells isolated from Hif-1αfl/fl Cre (wild-type [WT]) or Hif-1fl/fl Cre+ (Cre) mice that were administered tamoxifen. Two independent experiments were performed and the data presented are one representative experiment. (B) Population doublings (PD) of NIH3T3 cells expressing an inducible form of a constitutively activated HIF-1α. Two independent clones were cultured in the presence or absence of doxycycline. Data are a representative experiment performed in triplicate ± SD. Inset shows a Western blot of HIF-1α expression in the transfected cells. Cells were treated with 200 μM desferrioxamine (cntrl), or exposed to vehicle control (−), or 0.5 μg/mL doxycycline (+). The doxycycline induction of transfected HIF-1α is comparable to the level of induction of endogenous HIF-1α in response to desferrioxamine. Total Akt was used as a loading control.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
6.
Figure 2.

Figure 2. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

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.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
7.
Figure 4.

Figure 4. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

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.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.
8.
Figure 7.

Figure 7. From: The transcription factor HIF-1? plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis.

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.

Julian J. Lum, et al. Genes Dev. 2007 May 1;21(9):1037-1049.

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