Fig. 7.

Role of LKB1/AMPK tumor suppressor axis on DVL’s function on YAP. a The MCF-10A cells expressing tetracycline-inducible shRNA against DVL3 were cultured under sparse culture condition and treated with 2-deoxyglucose (2DG, 3 mM) and metformin (Met, 5 mM) for 16 h period in absence (−) or presence (+) of doxycycline (Dox). Endogenous YAP localization was determined by confocal microscopy. Inset, DAPI nuclear stain; Scale bar, 10 μm. b The wt and DVL-TKO 293 T-REx cells were treated with 2DG and Met, and nuclear YAP abundance was then determined by immunoblot analysis (left panels) and confocal microscopy (right panels). Inset, DAPI nuclear stain; Scale bar, 5 μm. c Kinase-dead GFP-fused AMPK or flag-tagged LKB1 mutant was co-transfected with control (−) or HA-tagged DVL3 (+) in 293 cells. Whole-cell lysates (WCL) were immunoblotted with indicated antibodies, and nuclear fractions were used for nuclear YAP abundance (left panels). The TEAD (right upper) or TCF/LEF (right lower) reporter was co-transfected with DVL in empty vector transfected cell (vector) or kinase-dead AMPK (AMPK-KD) or LKB mutant (LKB-KD), and the relative fold repression of reporter activity was measured from triplicate experiments (mean ± SD). d The wt and AMPKα1/α2 double-knockout (DKO) MEFs were stably transfected with inducible DVL3 and nuclear YAP abundance was examined by immunoblot analysis (left panels) and immunofluorescence (right panels) in the absence (−) or presence (+) of doxycycline for a 48 h period. Upper inset, DAPI nuclear stain; Lower inset, HA (DVL3); Scale bar, 5 μm. e The A549 cells were stably transfected with inducible DVL3, and nuclear YAP abundance and DVL3 localization under sparse or confluent culture condition were examined by immunoblot analysis (left panels) and immunofluorescence (right panels) in presence of doxycycline. Inset, DAPI nuclear stain; Scale bar, 5 μm. f The 293 cells (1 × 10⁶) were transiently transfected with YAP and DVL3 in combination with vector control or LKB-KD mutant, and the cells were inoculated into the flank of athymic nude mice (n = 10). The empty vector or LKB-KD transfected cells served as control. Tumor volume was measured 5 weeks end-point (Mann–Whitney test)