Fig 2: LKB1 degrades DBC1 independently of AMPK.A, 293T cells were transiently transfected with HA-AMPK (1.5 µg) and Flag-LKB1 (1.5 µg) constructs, and the cell lysates were blotted using indicated antibodies. C, 293T cells were transfected with HA-AMPK or Flag-LKB1 constructs as in A. In addition, they were also cotransfected with myc DBC1 (2 µg). At 48 h posttransfection, the cells were lysed and protein extracts were analyzed by Western blotting, using indicated antibodies (Flag-LKB1 band is indicated by black arrow). B and D, graphs representing relative DBC1 expression in the indicated control, AMPK, and LKB1 transfected 293T cells. The values indicate mean ± SEM; n = 3 biological replicates. Protein expression in control cells was arbitrarily taken as 1 in all of the Western blotting quantifications. E, DBC1 levels were not decreased by kinase dead (KD) mutant of LKB1. 293T cell lines were transfected with myc-DBC1, Flag-LKB1 (WT), and mutant of Flag-LKB1 (KD) constructs (2 µg each). Western blot of the lysates was performed using anti-myc for DBC1 and anti-Flag antibody for LKB1. F, effect of LKB1 over-expression on DBC1 amounts in stable cell lines. Lysates from U2OS and HeLa cells stably overexpressing LKB1 were blotted for endogenous DBC1 using anti-DBC1 antibody. G, LKB1 mediated down-regulation of DBC1 is independent of AMPK. U2OS cells stably expressing DBC1 were treated with different AMPK activators (AICAR (1 mM), 2-deoxy glucose (25 mM) and metformin (25 mM)) for 12 h. The lysates were blotted for DBC1 using anti-DBC1 antibody. H, U2OS cells were treated with different concentrations of okadaic acid (OA) (for 16 h) to inhibit PP2A. Western blot was carried out with DBC1, pLKB1, and LKB1 antibodies. I, U2OS cells stably expressing PyST (U2OS pTREX-PyST-HA-FLAG) were treated with different concentrations of NAD for 24 h. pLKB1 and LKB1 amounts were determined by Western blotting. J, 293T Cells were transiently transfected with myc-DBC1 (1 µg) and FLAG-LKB1 (1 µg) constructs. The cells were treated with 10 µM of MG132 for 5 h followed by Western blotting of the cell lysates. Proteasome inhibitor MG132 prevented LKB1-induced degradation of DBC1. DBC1, deleted in breast cancer 1; PyST, polyoma small T antigen.

Fig 3: LKB1 regulates AKT1 via DBC1 and TRB3.A and B, PyST inhibits phosphorylation of AKT1. Lysates from PyST-U2OS cell lines were blotted with antibodies specific for phosphorylation of S473 and T308 residues on AKT1. The membrane was stripped and blotted for total AKT protein levels. C, effect of DBC1 overexpression on AKT1 phosphorylation. Protein lysates from U2OS cells stably expressing DBC1 were blotted for AKT1 S473 phosphorylation. D, effect of LKB1 overexpression on AKT S473 and T308 phosphorylation levels in U2OS cell lines stably expressing LKB1. Proteins were detected with the indicated antibodies. E, 293T cells were transiently transfected with AKT1, LKB1, and AKT1/LKB1 constructs (1.5 µg each). pAKT 473, pAKT 308, total AKT, and LKB1 were detected in Western blotting using respective antibodies. F, 293T cells were transiently transfected with FLAG-FOXO3a (2 µg) alone or together with AKT1, LKB1, or DBC1 constructs (1.5 µg each). Cell lysates were blotted for pFOXO3a and total FOXO3a using indicated antibodies. Other transfected proteins like myc-DBC1, HA-AKT1, and Flag-LKB1 were also detected using indicated antibodies. GFP was used as transfection control and was detected with anti-GFP antibody. G, soft agar assay for U2OS cells stably over-expressing DBC1 was done in triplicates. Pictures of colonies as seen in the figure were taken after 3 weeks of growth. H, quantitative representation of the soft-agar assay. After 4 weeks of growth, the colonies were stained with iodonitrotetrazolium for overnight. After staining, colonies of 2 mm size or bigger were counted, and the data was analyzed by GraphPad Prism 8 (**p < 0.01). I, Western blot showing the expression of DBC1 in different cancer cell lines as indicated. DBC1, deleted in breast cancer 1; PyST, polyoma small T antigen.

Fig 4: Effect of resveratrol and caloric restriction on protein expressions of p53, FOXO3a, HuR, AROS and DBC1 in brains of aging ratsAll values were expressed as means ± SD. *P < 0.05, **P < 0.01 compared with negative control group; #P < 0.05, ##P < 0.01 compared with model negative group; ?P < 0.05 compared with CR group (RES group). NG, negative control group; MG, model control group; RESL, low dose of resveratrol group; RESH, high dose of resveratrol group; CR, caloric restriction group.

Fig 5: PySTdown-regulatesDBC1viaactivation of LKB1.A, schematic representation of different domains of DBC1 that interact with SIRT1 and PARP-1. B, detection of PARylation in PyST expressing cell lines using immunofluorescence. The cells with circles represent the ones in mitosis in control (-dox) and PyST expressing cells (+dox), (20× magnification; the scale bar represents 20 µm). C, one cell was chosen from the control (-dox) and PyST expressing cells (+dox) from the experiments, as shown in (B) and magnified to highlight the expression of PAR in mitosis. The magnified cells as shown in panel C are different images but from the same set of experiments (63× magnification; the scale bar represents 10 µm). D, 293T cells were transfected with myc DBC1 (1.0 µg), PyST-HA-Flag (1.0 µg), and Flag SIRT1 (1.0 µg), and Western blotting was performed with anti-acetyl lysine (Ac-K) antibody. E, 293T cells were transfected with PyST-HA-Flag (1.5 µg) and myc-DBC1 (1 µg) constructs. Immunoprecipitation was carried out with anti-DBC1 and anti-IgG (control) antibody, followed by Western blotting to confirm the interaction between PyST, DBC1, SIRT1, and PARP-1, using the indicated antibodies. F, Western blotting of a-PAR levels in the cell lysates obtained from PyST-U2OS cell line. G, U2OS cells stably over-expressing PyST were induced for 24 h. The cell lysates were blotted for pLKB1 (S428) and total LKB1. H, Western blot showing immunoprecipitation (IP) of PyST-HA-FLAG using anti-FLAG antibody and detection of DBC1, PP2A-C, HSP70, and LKB1 using the respective antibodies, as indicated. Please note that this experiment is the same as was carried out for Figure 2H. Although DBC1, PP2A-C, and FLAG-PyST were shown in Figure 2H, these bands (only for the control (Con) and PyST lanes) are reproduced here as they are part of the H also. This is indicated by the dotted line. The bands of LKB1 and HSP-70 of this experiment are shown here, below the dotted line. Similarly, for the “input” part of this experiment (right panel), the bands of DBC1, PP2A-C, Flag-PyST, and Actin are reproduced from Figure 2H (“Input”). These two parts (in Figs. 2H and 5H) are assembled again here for the sake of understanding and supporting the model in Figure 9. DBC1, deleted in breast cancer 1; HC, heavy chain; HSP, heat shock protein; PARylation, poly ADP ribosylation; PP2A, protein phosphatase 2A; PyST, polyoma small T antigen.