Fig 2: Pravastatin (PS)-induced AMPK activation is mediated by LKB1 in BM-EPCs under 24 h of Dex exposure. (A) Western blot analysis for the PS (1 µM)-induced autophagy activation in CaMKKß or LKB1 knock-downed BM-EPCs under Dex (1 µM) stress for 24 h. (B) The quantitative analysis demonstrated that PS-induced autophagy activation was abrogated in LKB1-siRNA BM-EPCs. Values are expressed as the mean ± SEM of three independent experiments. ##p < 0.01 vs. Dex.

Fig 3: Characterization of the farnesylation-deficient LKB1C433S/C433S mouse(A) The knockin construct, the endogenous LKB1 allele containing exons 1–10 and the target allele with the puromycin cassette (Puro) removed by Flp (flippase) recombinase are depicted. The black/grey rectangles represent exons and the black triangles represent FRT (flippase-recognition target) sites. TK, thymidine kinase. (B) To genotype mice, genomic DNA was PCR-amplified. Sizes are indicated in bp. The breeding strategy employed to generate LKB1C433S/C433S mice is shown with the number and percentage of each genotype obtained indicated. (C) Tissues lysates from wild-type LKB1+/+ and LKB1C433S/C433S mice and MEFs were subjected to immunoblotting using anti-LKB1 and anti-LKB1 farnesylation-specific (LKB1 Farn C433) antibodies. (D) Testis lysates from LKB1+/+ and LKB1C433S/C433S mice were immunoblotted with an anti-LKB1 antibody to detect the LKB1long (LKB1L) and LKB1short (LKB1s) isoforms. Band intensities were quantified using Li-Cor. Results are means±S.E.M. for four mice per genotype. (E) LKB1 was immunoprecipitated (IP) from the liver and muscle (EDL) from wild-type LKB1+/+ (+/+) and LKB1C433S/C433S (C433S/C433S) mice and the in vitro kinase activity towards the LKBtide peptide was measured. Immunoprecipitates were also immunoblotted (IB). Assays were performed in duplicate from tissues taken from six mice per genotype and the results are means±S.E.M. The broken line represents the background activity as measured with pre-immune IgG. (F) As (E), except that the immunoprecipitated LKB1 protein was used to activate a recombinant heterotrimeric AMPK complex (a1ß2?1) and then AMPK kinase activity towards the AMARA peptide was measured. Assays were performed in duplicate from tissues taken from six mice per genotype and the results are means±S.E.M. (G) Liver and MEFs from wild-type (WT) LKB1+/+ (+/+) and LKB1C433S/C433S (KI/KI) mice were submitted to subcellular fractionation. Cytoplasmic, membrane and nuclear fractions were immunoblotted with the indicated antibodies. Blots for two animals out of four per genotype are shown.

Fig 4: LKB1 regulates Tribbles 3 expression through DBC1.A, graphical representation of microarray data from control (-dox) and PyST expressing U2OS cells (+dox) for Tribbles 3 (TRB3). These experiments were carried out as biological triplicates. B, fold change for TRB3 mRNA in PyST expressing cells (+dox, 24 h) as compared to control cells (-dox), as shown by RT-qPCR. The data were normalized by using actin as control. C, PyST increases protein levels of TRB3. pTREX-PyST cells were induced for expression of PyST for different time periods as indicated (0 to 52 h), and cell lysates were then analyzed by Western blotting for DBC1, TRB3, and PyST using antibodies against respective proteins. D and E, confirmation of DBC1 expression in U2OS stable cells at protein and mRNA levels by Western blotting (using anti DBC1 antibody) and RT-qPCR, respectively. F and G, effect of the overexpression of DBC1 and LKB1 respectively, on TRB3 mRNA levels using RT-qPCR. For all these experiments, total cellular RNA was extracted from respective cell lines. After cDNA synthesis, RT-qPCR was carried out using appropriate primers and normalization was carried out using actin as control. The experiments were carried out in duplicates and the gene expression normalized to actin expression and fold change was calculated using ??Cq method. p-values were calculated using student’s t test. Shown are *p < 0.05, **p < 0.01, ***p < 0.001. The error bars represent mean ± SEM. DBC1, deleted in breast cancer 1; PyST, polyoma small T antigen.

Fig 5: ODInCas9 mouse non-small cell lung cancer models.a Schematic representation of generation of ODInCas9 KrasG12D;Trp53-/-, KrasG12D;Stk11-/-, and KrasG12C;Trp53-/- non-small cell lung cancer models. ODInCas9 mice are treated with doxycycline 3 days before administration via oral aspiration of model-specific AAV targeting either Kras by sgRNA and HDR and Trp53 or Stk11 by sgRNA. Doxycycline treatment is kept 1 day post viral administration. Lung adenocarcinoma kinetics is specific for each lung cancer model. b Representative HE staining of lung histology capturing the various stages of lung adenocarcinoma. c Western blots showing Trp53 and Stk11 expression in cell lines generated from either Kras;Trp53 or Kras;Stk11 lung tumors and d in multiple independent lung tumors from the Kras;Trp53 and Kras;Stk11 models. MC38, M2, HEK293 cell lines and non-tumor bearing lung shown as positive control. e Classification of amplicon sequencing of lung cancer tissue at Kras and Trp53 locus. f DNA in situ hybridization targeting KrasG12D. g Immunohistochemistry section of lung tumors staining for Ki67, pERK, pMEK, CD31, aSMA, F480, CD45R, CD4/8, pSPC, ?H2AX, Tenascin C, and Nkx2.1.