Fig 2: PP2A inhibitor recovers high fructose-induced dysfunction mediated by NO reduction. MVECs were pretreated with or without 20 nM OA for 1 h and then stimulated with fructose. A NO production from MVECs (after pretreatment with fructose for 24 h) (n = 4). Western blot analysis showing the protein expression of eNOS, P-eNOS (Ser1177) (B, C), PP2AC, and P-PP2A (Tyr307) (D, E) in MVECs (after pretreatment with fructose for 8 h) (n = 3). Relative protein levels of P-eNOS (Ser1177) were normalized to that of eNOS, and those of P-PP2A (Tyr307) were normalized to that of PP2AC. All data are presented as the mean ± SEM. C, control; Fru, fructose. *P < 0.05, **P < 0.01 when compared with the respective control groups

Fig 3: AngII induces YAP/TAZ activation and the expression of pro-inflammatory cytokines via protein phosphatase 2A C (PP2Ac) in HUVECs. (A) AngII increased PP2Ac activity; inhibition of PP2Ac with LB-100 suppressed AngII-mediated enhancement of PP2Ac activity in HUVECs. The protein expressions of PP2Ac (B) and p-PP2Ac (C); (D–G) Inhibition of PP2Ac with LB-100 suppressed AngII-induced YAP/TAZ nuclear translocation, the protein expressions of YAP (D) and TAZ (F) in the nucleus fraction, and the protein expressions of p-YAP (E) and p-TAZ (G) in the cytosolic fraction of HUVECs; protein expressions of proinflammatory cytokines MCP-1 (H) and TNFa. (I) *p < 0.05, vs. Ctr group, #p < 0.05, vs. AngII group.

Fig 4: 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).

Fig 5: CDC25B alters PP2A interaction with AMPK and subsequent AMPK phosphorylation. (A) BT549 and HS578T cells were transfected with Negative siRNA, siCDC25B, EV or CDC25B plasmids. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with control IgG or anti-AMPKa antibody. The immunoprecipitates were blotted with the indicated antibodies. (B) CDC25B knockout BT549 and stable knockdown HS578T cell lysates were subjected to IP with control IgG or anti-AMPKa antibody examining the AMPK–PP2A-C interaction. The immunoprecipitates were blotted with the indicated antibodies. (C) BT549 and HS578T cells were transfected with Negative siRNA or siAMPK a1and a2. Forty-eight hours later, cell lysates were subjected to IP with control IgG or anti-CDC25B antibody examining the CDC25B–PP2A interaction. The immunoprecipitates were blotted with the indicated antibodies. (D) BT549 and HS578T cells were transfected with negative siRNA (Neg) or CDC25B siRNA for 24 h, and then treated with 20 mM metformin and 2 µM LB100 for 48 h. Cells were harvested for immunoblotting of AMPK pathway. Data information: all data presented are a representation of N = 3 independent experiments.