Figure 4. AMPK-mediated Ser408 phosphorylation inhibits Gli1 driven MB cell proliferation.

(A) (Left) DAOY cells stably expressing Flag-Gli1 WT, S408A and S408E mutants were seeded on 6-well plates and grown for 2 weeks. Colonies (larger than 1,5 mm) were counted. Middle, representative images of the colony assay. (Right) The amounts of the expressed Gli1 proteins were analyzed by immunoblotting with Flag antibody. Tubulin, loading control. (B) Proliferation rate of DAOY stable clones. Each clone was seeded in triplicate and counted at the indicated times. (C) As in B), cells were treated with A-769662 (25 μM) and counted at the indicated time points. (D) Model of the AMPK/Gli1 mediated mechanism of Hedgehog pathway regulation. Under normal conditions, Gli1 binds to Hedgehog target genes promoters and activates their expression to regulate proliferative and developmental processes. Activation of AMPK by drugs or energy stress phosphorylates Gli1, leading to its degradation. With this mechanism, AMPK inhibits Hedgehog pathway and Hedgehog-dependent events. Results are expressed as mean and SD of three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 for the indicated comparisons.