Fig 2: 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.

Fig 3: CDC25B interacts directly with the PP2A-C subunit. (A) Top: GST-CDC25B was used to pull down purified His-PP2A-A, His-B56d, His-PP2A-C, His-PP2A-A + B56d, His-PP2A-A + PP2A-C, His-B56d + PP2A-C or His-PP2A-A + B56d + PP2A-C. Bottom: GST immunoblot of SDS-PAGE gel demonstrating the amount of CDC25B used for pull-down experiments. (B andC) The CDC25B-B56d interaction in Neg or PP2A-C KD cells. BT549 and HS578T cells were transfected with Neg or siPP2A-C. Forty-eight hours later, cell lysates were subjected to IP with control IgG or anti-CDC25B antibody examining the CDC25B–B56d interaction. Reciprocal IP with B56d antibody was also performed to examine the CDC25B–B56d interaction. The immunoprecipitates were blotted with the indicated antibodies. (D) B56d, not B55d, interacts with CDC25B. BT549 cells were transfected with siB56d. Forty-eight hours later, cell lysates were subjected to IP with control IgG or anti-CDC25B antibody examining the CDC25B–B55d interaction. Reciprocal IP with B55d antibody was also performed to exam the CDC25B–B55d interaction. The immunoprecipitates were blotted with the indicated antibodies. Western blot analysis demonstrates the protein level of B55d in Neg or B56d KD cells. Data information: all data presented are a representation of N = 3 independent experiments.

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 interacts with PP2A. (A) HEK-293T cells were transfected with EV or Flag-CDC25B plasmids for 24 h then treated with 10 mM metformin for 48 h. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The immunoprecipitates were blotted with the indicated antibodies. (B) HEK-293T cells were treated with 10 mM metformin treatment for 48 h. PP2A-C and CDC25B were detected using immunofluorescence. The nucleus was labeled by DAPI staining. Images were taken at 100× magnification. (C) BT549 and HS578T cell lysates were subjected to immunoprecipitation with control IgG or anti-CDC25B antibody. The immunoprecipitates were blotted with the indicated antibodies. (D) BT549 and HS578T cell lysates were subjected to immunoprecipitation with control IgG or anti-PP2A-C antibody. The immunoprecipitates were blotted with the indicated antibodies. (E) CDC25B knockout BT549 cells were transfected with EV, WT CDC25B (WT), CDC25B S353A, CDC25B S353E or CDC25B C488S constructs for 48 h. Cell lysates were subjected to immunoprecipitation with control IgG or anti-Flag antibody. The immunoprecipitates were blotted with the indicated antibodies. (F) BT549 cells were treated with 20 mM metformin for 48 h. Cell lysates were subjected to immunoprecipitation with control IgG or anti-CDC25B antibody. The immunoprecipitates were blotted with the indicated antibodies. (G) CDC25B knockout BT549 cells were transfected with EV, FL CDC25B, CDC25B N-terminal domain (1–373 aa, NTD) or CDC25B C-terminal domain (374–580 aa, CTD) for 48 h. Cell lysates were subjected to immunoprecipitation with control IgG or anti-Flag antibody. The immunoprecipitates were blotted with the indicated antibodies. Data information: all data presented are a representation of N = 3 independent experiments.