Fig 2: CDC25B modulation of metformin cytotoxicity via PP2A-AMPK. (A) BT549 and HS578T cells were transfected with EV, CDC25B or PP2A-C plasmids, negative siRNA or siAMPKa1 and a2, and then treated with increasing doses of metformin for 72 h, and cell survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. Knockdown or overexpression efficiency is shown in mRNA levels. (B) CDC25B knockdown BT549 and HS578T were transfected with siPP2A, and then treated with increasing doses of metformin for 72 h, and cell survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. Knockdown efficiency is shown in mRNA levels. (C) CDC25B knockout BT549 and stable knockdown HS578T cells transfected with EV, full length CDC25B (FL), CDC25B N-terminal domain (1–375 aa) (NTD), or CDC25B C-terminal domain (374–580 aa) (CTD), were treated with increasing doses of metformin for 72 h and cell survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. (D) CDC25B knockout BT549 and stable knockdown HS578T cells transfected with EV, WT CDC25B (WT), CDC25B S353A, CDC25B S353E, or CDC25B C488S constructs, were treated with increasing doses of metformin for 72 h and cell survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. (E) Cells were transfected with indicated siRNA, and then treated with increasing doses of metformin for 72 h, and cell survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. Data information: all data presented are in the format of mean ± SEM of N = 3 independent experiments with three biological replicates for each experiment. Statistically significant differences were determined using the two-way ANOVA plus Tukey (**P < 0.01), ns represents not significant.

Fig 3: Vasodilation function unchanged in the PP2A cKO mice under high fructose stimulation. A Identification of the PP2ACa cKO mice by genotyping. In each group of experiments, the aortic rings were stimulated by fructose (0.25 mM and 2 mM) for 0.5 h, precontracted by using 10–6 M PE, and relaxed by 10–9–10–5 M Ach or SNP. Wire myograph recording the effect of fructose in concentration–response curves to the endothelium-dependent dilator Ach (B) and the endothelium-independent dilator SNP (C) in aortic rings from the PP2ACaflox/flox and cKO mice. D, E The maximal response of Ach and SPN. All data are presented as the mean ± SEM (n = 5 aortic rings). C, control; Fru, fructose; Ach, acetylcholine; SNP, sodium nitroprusside; PE, phenylephrine. *P < 0. 05, **P < 0. 01 when compared with the respective control groups

Fig 4: Pharmacological-mediated perturbation of the LCMT1-PME-1 methylation equilibrium leads to misregulation of spindle size and cell division. (A) Pharmacological inhibition of PME-1 with AMZ30 compared to DMSO vehicle control. Note that the levels of demethylated PP2AC decreased in cells treated with AMZ30. (B) Immunofluorescence microcopy showing that AMZ30-treated cells have abnormally short spindles. Bar = 5 µm. (C) Quantification of the percentage of cells in mitosis showing that inhibition of PME-1 with AMZ30 leads to a major increase in cells arrested in mitosis. (D) Quantification showing that treatment of cells with AMZ30 leads to a major increase in the percentage of mitotic cells with abnormal spindles (short, long, multipolar). (E) Quantification showing that treatment of cells with AMZ30 leads to a major increase in the percentage of unaligned chromosomes. (F) Quantification of metaphase spindle PTP distance (in µm) showing that AMZ30-treated cells have abnormally short spindles. (G) Quantification of metaphase spindle width (in µm) showing that AMZ30-treated cells have abnormally short spindle widths. (H) Quantification of metaphase spindle volume (in µm3) showing that treatment with AMZ30 leads to a decrease in volume. (I) Quantification of the total fluorescence intensity of mitotic spindle microtubules for AMZ30-treated cells, in arbitrary units (A.U.), showing that AMZ30 treatment leads to a decrease in total microtubule polymer. (C–I) Data represents average ± SDs of 3 independent experiments. ** = P < 0.005, *** = P < 0.0005.

Fig 5: CDC25B–PP2A regulates breast cancer patients derived 3D organoids’ response to metformin. (A) Organoids transfected with siAMPK, and then treated with 20 mM metformin for 6 days. Cell proliferation was monitored every 2 days. (B) Organoids transfected with siCDC25B (KD) were transfected with EV, WT CDC25B (WT), CDC25B S353E or CDC25B C488S constructs, and then treated with increasing doses of metformin for 72 h, and organoid survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. Organoid lysates were blotted with the indicated antibodies. (C) Organoids were transfected with siPP2A-C, and the growth was monitored every 2 days. (D) Organoids transfected with siPP2A-C were treated with increasing doses of metformin for 72 h, and organoid survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. Knockdown efficiency is shown in western blots. (E) Organoids were treated increasing doses of metformin alone or in combination with 2 µM of LB100 for 72 h, and organoid survival was then determined. The x-axis indicates drug dose, and the y-axis indicates the survival fraction after metformin exposure. (F) A simplified model depicting how CDC25B might regulate AMPK activity. Data information: all data presented are in the format of mean ± SEM of N = 3 independent experiments with three biological replicates for each experiment. For (A and B), statistically significant differences were determined using the two-way ANOVA plus Tukey (**P < 0.01), ns represents not significant. For (C, D and E), statistically significant differences were determined using the two-way ANOVA (**P < 0.01).