Fig 2: Quantitative PCR analyses of the relative levels of six AMPK subunits in ovarian tissues. Except for AMPK-α1, the boxplots indicate that statistically significant over-expressions of the other five AMPK subunits were detected in ovarian carcinomas compared with the normal. Borderline tumors were excluded from the analysis because of the small sample size. The abbreviations are A1 = AMPK-α1; A2 = AMPK-α2; B1 = AMPK-β1; B2 = AMPK-β2; G1 = AMPK-γ1; G2 = AMPK-γ2; Ca = Cancer; Normal N = 48; Borderline N = 5; Cancer N = 89.

Fig 3: PRMT1-mediated meR342-EZH2 enhanced PRC2 assembly through inhibiting AMPK-mediated pT311-EZH2.A Western blotting detecting EZH2 binding with meR342-EZH2, AMPKa1, SUZ12, EED after Co-IP Flag-EZH2 in MCF7-Flag-EZH2-WT and MCF7-Flag-EZH2-R342K cells. B, C Detection of pT311-EZH2 expression after knockdown PRMT1 in MDA-MB-231 (B) and MCF7 (C) cells. D, E Immunoblot analysed pT311-EZH2 expression in MCF7-Vector and MCF7-PRMT1 cells (D), or MDA-MB-231-Vector, MDA-MB-231-EZH2-WT and MDA-MB-231-EZH2-R342K cells (E).

Fig 4: Effect of deletion of CaMKKß on Thr172 and ACC phosphorylation in A549 cells.The CRISPR–Cas 9 system was used to delete CaMKKß in A549 cells (which lack endogenous LKB1 expression). (A) Western blot analysis of the parental A549 cells and the CaMKKß KO cell line with antibodies against ACC, AMPKa1, a2, pan-a, pan-AMPKß, CaMKKß and LKB1. For comparison, HEK293T cell lysate is included. Vinculin expression is shown as a total protein loading control. In all cases, duplicate samples from independent cell preparations are analysed. (B) Nucleotide content of prechloric acid extracts of parental or CaMKKß KO A549 cells, incubated with or without 12 mM 2DG for 60 min, was determined by capillary electrophoresis. In each case, a representative trace showing UV absorbance at 254 nm is shown and the migration of adenine nucleotide standards is indicated on the traces. (C) Parental and CaMKKß KO A549 cells were treated with 991 (5 µM), 2DG (12 mM) or both 991 and 2DG, for 60 min. Cell lysates were resolved by capillary electrophoresis and the levels of ACC and Thr172 phosphorylation, together with AMPK ß-subunit expression, were determined. In each case, a representative blot with two independent samples is shown. (D) Quantification of the blots shown in (C) determined by chemiluminescence. Results are shown as mean ± SEM for three independent experiments and are plotted as arbitrary units (AU).

Fig 5: GSTA1 overexpression decreased liver cancer cell proliferation, migration and invasion abilities. A. CCK8 assay showed that GSTA1-transfected cells had decreased cell viability compared with empty vector control cells (*P < 0.05). B. Colony size and density in GSTA1 groups were smaller and rarer than those in control groups (*P < 0.05). The abilities of migration (C.) and invasion (D.) of liver cancer cells transfected with GSTA1 were decreased, which were detected by trans-well assays (*P < 0.05). Representative images were selected randomly from 5 fields (crystal violet staining, 400×) in each group and quantified by mean of five random fields. E. GSTA1 overexpression downregulated AMPK/mTOR. Western blot showed that GSTA1 protein was overexpressed in GSTA1-transfected liver cancer cells, indicating a successful transfection. In 97H and SNU449 cells, GSTA1 overexpression increased LKB1 and p-AMPK Thr172 protein expression while the total AMPK amount remained unchanged. Western blotting results indicated that p-mTOR 2448 decreased in cells overexpressing GSTA1, compared with controls. Besides, p-p70 S6K as well as MMP-9 were downregulated in the GSTA1 groups.