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. 2016 Jan 29;7(8):9538–9549. doi: 10.18632/oncotarget.7070

Figure 1. AMPK inhibits Hh signaling only in human cells.

Figure 1

(A) NIH3T3 (left) and human DAOY (right) cells were treated with Sag or Dmso, AICAR (2 mM), 2-deoxyglucose (2DG, 25 mM) or Metformin (5 mM) as indicated, for 6 hours. Levels of Gli1 mRNA were analyzed by quantitative PCR. (B) Wild type or AMPK−/− MEF cells were treated with Sag or Dmso, AICAR (2 mM), 2-deoxyglucose (2DG, 25 mM) and Metformin (5 mM) as indicated for 6 hours. Gli1 mRNA levels were evaluated by quantitative PCR. The experiment was repeated three times. (C) NIH3T3 (top) and DAOY cells (bottom) were treated with Sag or Dmso, and with A-769662 (25 μM) for 24 hours. Gli1 mRNA levels were evaluated by quantitative PCR. (D) (Top) Quantitative real time PCR on AMPK-deficient DAOY cells. DAOY cells were infected with lentiviruses expressing shRNA targeting both α1 and α2 AMPK subunits (shAMPK) or with non-targeting shRNA. Cells were treated with Sag or Dmso and A-769662 (25 μM) for 24 hours as indicated. Gli1 mRNA levels were assessed. Knockdown efficiency was analyzed by immunoblotting with the indicated antibodies (bottom). (E) DAOY cells were transfected with siRNA against Sufu (siSufu), or with non-targeting siRNA (siCtr); cells were then treated with A-769662 (25 μM) or Dmso and Gli1 mRNA levels were measured by quantitative real time PCR. Results are expressed as mean and SD of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 and ns (not significant) for the indicated comparisons.