Fig 2: WT and mutant LKB1K78I exert significant tumor suppressive activity. (A) Western blot analysis of LKB1 protein expression in a normal prostate epithelial cell line (PWR1E) and in a panel of PCa cell lines (technical duplicates are shown). Representative image from three independent experiments is shown. (B) Western blot analysis of LKB1 protein expression in DU145 cell line with ectopic expression of WT Flag-LKB1 (LKB1WT), the Flag-LKB1-K78I mutant (LKB1K78I), or transduced with mock vector (pBABE) using 22RV1 cells as a positive control (n = 3). Representative image from three independent experiments is shown. (C–E) Quantification (left) and representative image (right) of anchorage-independent growth (C; n = 8, independent experiments), migration (D; n = 6, independent experiments), and invasive growth (E; n = 5, independent experiments). Black circle highlights the original size of the spheroid and dashed line highlights the size of the spheroid after 48 h of incubation in DU145 cells with ectopic expression of LKB1WT or LKB1K78I or transduced with mock (pBABE). Data are normalized to control (pBABE; dashed line). (F and G) Effect of LKB1 reconstitution in DU145 cells with ectopic expression of LKB1WT or LKB1K78I or transduced with mock (pBABE) on tumor formation (F) and tumor weight (G; left). n = 13–14 tumors per condition. (H and I) Evaluation of metastatic capacity of control (pBABE) and LKB1WT- and LKB1K78I-expressing DU145 cells using intracardiac xenotransplant assays. Luciferase-based signal intensity of DU145 cells in limbs was monitored in vivo (H; n as indicated) and ex vivo (I; mean signal of both tibia was calculated and presented; n as indicated in the dot plot) for ≤38 d. Representative luciferase images are presented referred to the quantification plots. Statistic tests: one-sample t test (C–E), one-tailed unpaired Student’s t test (F and G), one-tailed Mann–Whitney U test (H and I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent SEM.

Fig 3: LKB1 resupply suppresses tumor formation induced by depletion of RhoBTB2. (A) Confocal images of wing discs showing the effect of LKB1 depletion. GFP (green), DAPI (blue) and Dlg (red). Genotypes are indicated in the figure. All images correspond to the same scale. (B) Representative images of wing discs showing the influence of wildtype LKB1 overexpression respectively kinase-inactive LKB1 expression on the RhoBTB2 depletion phenotype. Discs were stained for actin. Genotypes are indicated. All images were taken at the same magnification. (C) Graphs showing the effect of wildtype LKB1 (LKB1WT) overexpression respectively kinase-inactive LKB1 (LKB1KI) expression on the frequency of wing disc tumor formation. Data are presented as the number of tumor-bearing larvae scored per day ± SD p-values were determined using a non-parametric Mann-Whitney test.

Fig 4: Changes physiological and Nur77-AMPK related proteins levels in mice fed a normal diet (NC) and a high-fat diet (HFD). (A) Oil O red staining (40× magnification); and (B) hematoxylin and eosin (H&E) staining (40× magnification) of liver tissue of in mice fed NC and HFD. Left, mice fed a NC (10% calories from fat). Right, mice fed a HFD (60% calories from fat). Sections of the liver of all mice were prepared after they were fed the respective diets for 8 weeks. (C) Weekly weight change in mice of the NC and HFD groups. (D) Indicators of serum associated liver injury in mice. Results of the t-test showed statistical differences AST, TBA, cholesterol. ALT and TG showed no significant changes. (E) Expression of metabolism-associated proteins in NC and HFD groups. (F) Results of the t-test showed statistical differences in the expression of relevant indicators. Nur77, HMGCR, p-ACC, and CPT1A were evaluated. A decline in expression was observed in LKB1, AMPKα. Acetyl-CoA carboxylase (ACC) showed no significant change. *P < 0.05, **P < 0.01, ***P < 0.001, t-test.

Fig 5: Ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl] acetate (TMPA) interfered with FFAs stimulation in HepG2 cells and primary hepatocytes. The combination of Nur77 with LKB1 in the nucleus inhibited the phosphorylation of LKB1 and AMPKα. The stability of LKB1-Nur77 was compromised under the influence of TMPA. LKB1 was subsequently translocated to the cytosol from the nucleus and activated AMPKα. The phosphorylation of AMPKα suppressed ACC by phosphorylation and activated CPT1A, the rate-limiting enzyme of lipid oxidation.