Transcriptional Regulation of LKB1 Figure 8. NF-Ya transcription factor is required for LKB1 gene expression. Knockdown of endogenous NF-Ya by siRNA inhibits LKB1 expression in ‘‘444’’, C33a and IMR-90 cells. Cells were transfected either with scrambled siRNA (Ctr) or with a siRNA targeting NF-Ya (NF-YA). Total RNA was purified and mRNA levels of NF-YA, NF-YB, LKB1 and GAPDH were analysed by RT-PCR using ethidium bromide staining (A). Equal amounts of total protein (5 mg) were separated by SDS-PAGE and analysed by western blotting using antibodies against LKB1, phospho- AMPK (Thr172) and actin (B). The data shown is representative of three independent experiments. doi:10.1371/journal.pone.0032590.g008 The 59-flanking region of the LKB1 coding sequence encom- passing nucleotides 21536 to +1321 relative to the transcription start site was amplified by PCR using 200 nM of forward (59- CACCCTGCCTAATGTCCCTA-39) and reverse (59-GAGTC- CAGCACCTCCTTCAC-39) primers, 2 ng/ml of genomic DNA from ‘‘444’’ cells as a template and 0.05 U/ml of PfuUltra HF DNA polymerase (Stratagene) in a final volume of 25 ml PfuUltra reaction buffer containing 0.1 mM dNTP’s, 4% of dimethylsulf- oxide and 2% of formamide. The PCR-product was purified, cloned into the pJET1.2 plasmid using the CloneJET PCR Cloning Kit (Fermentas) according to the manufacturer’s instruc- tions and verified by DNA sequencing. The LKB1 promoter (position 21536 to +727) was then subcloned into the SmaI site of the pGL3-Basic luciferase reporter vector by PCR amplification using the same forward primer and the reverse primer (59- Figure 9. FOXO transcription factors are required for LKB1 gene expression. Knockdown of endogenous FOXO3 and FOXO4 in ‘‘444’’, C33a and IMR-90 cells. Cells were transfected either with scrambled siRNA (Ctr) or with a siRNA targeting all FOXO family members (FOXO). Total RNA was purified and relative mRNA levels of FOXO3, FOXO4, LKB1 and GAPDH were analysed by RT-PCR using ethidium bromide staining (A). Equal amounts of total protein (5 mg) were separated by SDS-PAGE and analysed by western blotting using antibodies against FOXO3, LKB1 phospho-AMPK (Thr172) and actin (B). The data shown is representative of three independent experiments. doi:10.1371/journal.pone.0032590.g009 GCCCACGGACAAGTATGAAC-39) with Phusion High-Fideli- ty DNA-polymerase (Finnzymes) according to the manufacturer’s instructions. Deletion mutants were generated by PCR using primers listed in Table S1 and inserted into the SmaI site of the pGL3-Basic plasmid. Substitution mutants of the LKB1 promoter were derived from the deletion mutants by inserting a second PCR product expanding from 2549 to the deletion end point, using an upstream primer containing a SacI site and the 2549 sequence and downstream primers containing a NheI site and the sequence adjacent to the respective deletion end point (Table S1). PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e32590