Skip to main content
. 2021 May 17;118(21):e2002486118. doi: 10.1073/pnas.2002486118

Fig. 5.

Fig. 5.

PI5P4K inhibition reduces tumor cell survival. (A) CC260 (0, 5, and 10 μM) caused AMPK activation and mTORC1 inhibition in BT474 cells after overnight treatment in DMEM without serum (n = 4). To measure glucose consumption and lactate production, BT474 cells were cultured in DMEM without serum for 24 h (the medium did not contain pyruvate). In DMSO control, glucose concentration decreased by 7.0 mM, and the final lactate concentration was 11.9 mM (∼85% of the consumed glucose was used for lactate fermentation). With 10 μM CC260, glucose decreased by 7.0 mM while lactate accumulated to 13.8 mM (∼99% used for fermentation) (n = 3). Mitochondrial and glycolytic ATP production rates were shown as means ± SEM (n = 16). (B) Simultaneous knockdown of PI5P4Kα and PI5P4Kβ by siRNA caused AMPK activation. Oligo “siControl” is of random sequence. After 1 d of transfection, BT474 cells were transferred to an siRNA-free DMEM with 10% fetal bovine serum and cultured for 2 more days before Western blot analysis (n = 4). CC260 did not cause further AMPK activation when PI5P4Kα and PI5P4Kβ were both knocked down by siRNA (n = 3). After transfection, BT474 cells were treated with 10 μM CC260 overnight before Western blot analysis. (C) AMPK activation was abrogated in BT474 cells expressing a refractory PI5P4Kβ double mutant. Cells were treated with compound (0, 5, and 10 μM) overnight in serum-free DMEM. (Left) Comparing the in vitro activities of wild-type and mutant PI5P4Kβ at different inhibitor concentrations (n = 3). (D) CC260 caused AMPK activation in both p53+/+ and p53−/− MCF-10A cells. (E) CC260 or glucose starvation (12 h) was selectively toxic toward p53−/− cells, causing a greater reduction of the number of proliferative cells in the clonogenic assay. The area covered by the cell was quantified, and the percentage decrease was based on comparison with p53+/+ and p53−/− cells without compound treatment or glucose starvation, respectively (n = 3). (F) After overnight CC260 (10 μM) treatment or glucose starvation in serum-free DMEM/F12 medium supplemented with 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin, the dead cells were measured by propidium iodide staining (n = 6). Results are shown as means ± SEM, * indicates P ≤ 0.01, ** indicates P ≤ 0.001, *** indicates P ≤ 0.0001, and # indicates P ≤ 0.05 based on Student’s t test. N.S., nonstatistically significant.