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. 2017 Mar 21;18(5):809–825. doi: 10.15252/embr.201643617

Figure 3. CUEDC2 regulates GLUT3 expression and aerobic glycolysis through GR .

Figure 3

  • A
    Lysates from PLC cells overexpressing HA‐CUEDC2 and FLAG‐GR were used for immunoprecipitation with control IgG, anti‐FLAG (left panel) or anti‐HA (right panel), followed by blotting with anti‐HA or anti‐FLAG.
  • B
    Protein levels of CUEDC2 and GR were detected by Western blot in PLC cells stably expressing shCUEDC2s in pSUPER or pLKO vectors or overexpressing CUEDC2.
  • C
    GR protein levels were detected by Western blot in PLC cells expressing shCUEDC2 in the presence or absence of proteasome inhibitor MG132 (5 μmol).
  • D
    HA‐Ub, FLAG‐GR, and shCUEDC2 cotransfected HEK293T cells were treated with proteasome inhibitor MG132 (5 μmol) for 6 h before lysis. Equal amount of proteins were used for immunoprecipitation with anti‐FLAG antibody, followed by blotting with anti‐HA or anti‐GR.
  • E
    Cellular localization of GR in CUEDC2 knockdown cells and NTC cells was analyzed by immunofluorescence staining with GR antibody. The nucleus was stained by DAPI. Scale bars, 10 μm.
  • F
    mRNA and protein levels of GLUT3 were determined by qRT–PCR and Western blot, respectively, in PLC cells stably expressing HA‐CUEDC2 with further knockdown of GR by shRNAs.
  • G
    A diagram shows the sites and sequences of potential glucocorticoid responsive elements (GREs) in GLUT3 gene.
  • H
    ChIP (left panel) and luciferase assays (right panel) were performed to identify GREs in GLUT3 gene.
  • I
    A diagram shows CUEDC2 enhances GLUT3 mRNA transcription by accumulating its transcriptional factor GR protein.
  • J–L
    Cellular glucose uptake and lactate production (J), cell growth curve (K), and apoptosis (L) were determined in PLC cells stably overexpressing CUEDC2 with further knockdown of GR by shRNAs.
Data information: (F, H, J–L) Data are presented as mean (± SD); n = 3 in each group. *P < 0.05 as compared to EV + NTC group in (F, J–L), to IgG group in (H, left panel) and to EV group in (H, right panel), respectively. P was calculated by Student's t‐test. β‐Actin served as loading control. NS: Not significant between indicated groups. Source data are available online for this figure.