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Fig. 1. Lkb1 modulates TGFβ signaling. (A) Fold induction of luciferase activity in MEFs with the indicated Lkb1 genotypes following infection with AdCre or AdLacZ (control) recombinant adenoviruses and subsequently transfected with either (CAGA)₁₂-luciferase, ARE-luciferase or BRE-luciferase plasmids and RL-TK to correct for transfection efficiency. Fold induction is indicated relative to control samples and all bars represent averages of three experiments performed with independently isolated MEFs using triplicate samples, except for the (CAGA)₁₂-luc Lkb1^lox/+ and (CAGA)₁₂-luc Lkb1^lox/lox bars, which represent averages from ten and five experiments, respectively. Right panel shows similar analysis with MEFs transfected with non-targeting (control) or Lkb1-targeting (siLkb1) siRNAs and (CAGA)₁₂-luc. Bars represent averages from two experiments using four to ten samples per experiment. Error bars indicate s.d.; ^**P<0.01, ^***P<0.001. (B) Western blotting analysis of Lkb1, activated Smad2 (Smad2-P, shown as p-Smad2) and total Smad2 from serum-starved (0.2% FCS) untreated (–) or TGFβ-treated (+; 1 ng/ml) control (AdLacZ-infected Lkb1^lox/– MEFs) or Lkb1^–/– (AdCre infected Lkb1^lox/– MEFs) MEFs. A background band in the Smad2-P blot is indicated with an asterisk. (C) Immunofluorescence staining using an antibody recognizing Smad2 and Smad3 of serum-starved (0% FCS, 22 hours) and subsequently untreated (–) or TGFβ-treated (1 ng/ml) control and Lkb1^–/– MEFs. The apparently lower total intensity of signal might be due to more diffuse localization of Smad2 and Smad3 in the cytoplasm of Lkb1^–/– MEFs compared with controls because no difference in total levels were noted in western blotting (see Fig. 1B). Nuclei are visualized with Hoechst. A single-cell magnification is shown in the inset. (D) (CAGA)₁₂-luc activity in control and Lkb1^–/– MEFs transfected with constitutively active ALK5 (ca-ALK5) or vector control and treated with added TGFβ as indicated. The value of relative (CAGA)₁₂-luc activity represents the ratio of (CAGA)₁₂-luc to Renilla-luciferase–thymidine-kinase (RL-TK), with the average from a representative experiment out of three using triplicate samples shown; error bars indicate s.d.

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