Development of an Lkb1 ASKA knockin mouse. (A, left) Lkb1 was stably knocked down in MDCK cells using a retroviral shRNA system. Cell lysates were blotted with antibodies to Lkb1 and actin. (middle) The tight junction marker ZO-1, apical marker gp135, and basolateral marker gp58 are properly localized in control (WT) and Lkb1 knockdown (KD) cells grown as monolayers. (right) The cortical actin network and apical marker gp135 are also properly localized in MDCK spheroids when the cells were grown in 3D Matrigel cultures. Bars, 20 µm. (B) A targeting vector was generated carrying the M129G mutation in exon 3 of Stk11 genomic DNA and a neomycin cassette flanked by two LoxP sites. The final targeted allele contained the M129G mutation and an adjacent intron with a single LoxP site. (C) Hematoxylin-eosin–stained sections of E14.5 Lkb1^WT/WT (left) and Lkb1^MG/MG (right) littermate embryos showing intact organogenesis. (D) Lkb1^MG protein levels are stabilized and kinase activity is inhibited by 1NMPP1 in MEFs. MEFs from Lkb1^WT/WT (lanes 1–4) and Lkb1^MG/MG (lanes 5–8) embryos were incubated for 16 h with DMSO (lanes 1, 2, 5, and 6) or 1 µM 1NMPP1 (lanes 3, 4, 7, and 8), and then treated with or without 2 mM AICAR for 1 h before collecting cell lysates. Western blots were analyzed with antibodies to Phospo-AMPKα (Thr172), AMPKα, and Lkb1. (E) Lkb1^MG kinase activity in embryonic explants is inhibited by 1NMPP1. Duplicate embyronic explants (pancreas; see Fig. 3) from Lkb1^WT/WT (lanes 1–4) and Lkb1^MG/MG (lanes 5–8) embryos were cultured in vitro on transwell filters and incubated 2 h with DMSO (lanes 1, 2, 5, and 6) or 1 µM 1NMPP1 (lanes 3, 4, 7, and 8) before collecting lysates. Western blots were analyzed with antibodies to Phospho-ACC(Ser79), ACC, Phospo-AMPKα(Thr172), AMPKα, and Lkb1.