Blood, Vol. 115, Issue 23, 4742-4749, June 10, 2010

Toll-like receptor–induced changes in glycolytic metabolism regulate dendritic cell activation
Blood Krawczyk et al. 115: 4742

Supplemental materials for: Krawczyk et al

Files in this Data Supplement:

• Figure S1. GLUT1 expression, measured by flow cytometry, in DCs following stiumulation with PS. PA or CpG (JPG, 137 KB) -
  Histograms indicate surface expression of GLUT1 on stimulated (black) or unstimulated (gray) DCs.
  
  
  
  
  
  
• Figure S2. TNFα production by DCs in the presence (black) or absence (grey) of glucose (JPG, 45.2 KB) -
  Cytokine levels were determined by ELISA 18 h following culture with LPS or PA.
  
  
  
  
  
  
• Figure S3. CD86 expression and production of p40 by DCs in the presence (+Glc) or absence (−Glc) of glucose, measured by intracellular p40 staining/surface CD86 staining and flow cytometry following 5 h of culture without (−) or with LPS (JPG, 147 KB) -
  Numbers represent % of gated cells.
  
  
  
  
  
  
• Figure S4. Activation of DCs in differing concentrations of glucose (JPG, 171 KB) -
  Flow cytometry was used to measure surface expression of CD40 following stimulation with LPS or PA. Numbers represent % of cells staining positive.
  
  
  
  
  
  
• Figure S5. LPS-induced activation of DCs in the presence of the glycolytic inhibitor 2-deoxyglucose (2-DG) (JPG, 156 KB) -
  Flow cytometry was used to measure surface expression of CD86 and MHCII (IAb) following LPS stimulation in the presence of the indicated concentrations of 2-DG. Unstimulated cells in the absence of 2-DG are shown for comparison (−). Numbers represent % of cells staining positive.
  
  
  
  
  
  
• Figure S6. AMPK expression is effectively suppressed by AMPK shRNAs (JPG, 357 KB) -
  AMPK expression measured by Western blotting with antibodies against AMPKα or pAMPKα (T172), in mouse embryonic fibroblasts transduced with a control hairpin or with a hairpin targeting AMPK (as described in the Materials and Methods). An antibody against actin was used to control for loading. As an additional control for knockdown, cells were stimulated with varying concentrations of 2-DG, which induces AMPK phosphorylation (as shown).
  
  
  
  
  
  
• Figure S7. LPS-induced production of TNF-α by IL-10^−∕− DCs is inhibited by exogenous IL-10 and 2-DG (JPG, 83 KB) -
  Cytokine levels in 18 h supernatants were measured by ELISA. Unstimulated cells (−) did not make measurable levels of cytkine. Data are mean value of replicates. Error bars represent standard deviations.
  
  
  
  
  
  
• Figure S8. 2-DG and AICAR inhibit LPS-induced IL-10 production (JPG, 100 KB) -
  Cytokine levels in 18 h supernatants were measured by ELISA. Unstimulated cells (−) did not make measurable levels of cytkine. Data are mean value of replicates. Error bars represent standard deviations.