Figure 1.
SESN2 interacts and regulates AMPKα1β1γ1 activity in MCF7 cells.
(A) MCF7 cells were plated into a 10 cm dish and grown until fully confluent. The cells were then lysed with lysis buffer and immunoprecipitation was preformed with the indicated AMPK subunit antibodies. These samples were then subjected to western blotting with AMPK subunit antibodies. A representative immunoblot from 3 independent experiments is shown. (B) Cells were transfected with 1 µg empty Flag vector (−) or 1 µg Sesn2F (+). Forty eight hours later the cells were lysed and immunoprecipitation was preformed with an anti-Flag antibody. The samples were then subjected to western blotting with the indicated antibodies. (C) MCF7 cells were treated with 1 µg empty Flag vector (−) or 1 µg Sesn2F (+). Forty eight hours later the cells were lysed and subjected to western blotting with the indicated phosphorylated AMPK antibodies. (D) The results from (C) were quantitated and expressed as the mean and SE from 3 independent experiments (* = P<0.05 and ** = P<0.01 compared to control).
Figure 2.
SESN2 is found in close proximity with active AMPK in the cytoplasm of MCF7 cells.
(A) Untreated MCF7 cells were fixed and stained with DAPI (blue), SESN2 (green), or P-AMPKα (red). (B) MCF7 cells were transfected with 1 µg Sesn2F vector (Sesn2F) for 24 h, followed by fixation and staining with DAPI (blue), SESN2 (green), or P-AMPK (red). The cells were then imaged at 40× and merged images are displayed on the right. Representative images from 3 independent experiments are shown.
Figure 3.
IR-induced expression and activity of AMPK is dependent on SESN2.
(A) MCF7 cells were treated with SESN2 siRNA for 48 h prior to being exposed to a single dose of 8Gy IR. The cells were lysed 24 h after IR and western blotting was preformed with the indicated antibodies. A representative immunoblot from 3–4 independent experiments is shown. (B) The results from (A) were quantitated and expressed as the mean and SE from 4 independent experiments (* = P<0.05 compared to control, ** = P<0.01 compared to control, # = P<0.05 compared to 8Gy IR).
Figure 4.
SESN2 in combination with IR modulates pathways of pro-survival and inhibits cell proliferation.
(A) MCF7 Tet-OFF SESN2 cells were incubated in the presence (+) or absence (−) of Dox-containing medium for 24 h before exposure to 8Gy IR. Twenty four hours later the cells were lysed and subjected to western blotting with the indicated antibodies. A representative immunoblot from 3 independent experiments is shown. (B) The results from (A) were quantitated and expressed as the mean and SE from 3 independent experiments (* = P<0.05 compared to control and # = P<0.05 compared to 8Gy IR). (C) MCF7 Tet-OFF SESN2 cells were incubated in the presence (basal) or absence (SESN2) of Dox-containing medium for 24 h before exposure to the indicated doses of IR. Seven days later the cells were fixed and stained with mythelene blue and the clonogenic survival was calculated. Results from 3 independent experiments were averaged and presented as the mean and SE (* = P<0.05 and ** = P<0.01 compared to the corresponding IR treatment alone) and plotted on a logarithmic scale using the linear quadratic equation. The results were normalized so that both the untreated (basal) and SESN2 overexpressing cells (SESN2) start at the same point. (D) MCF7 Tet-OFF SESN2 cells were incubated in the presence (control) or absence (S2+) of Dox-containing medium and treated with or without 1 µM compound C (CC) for 24 h before exposure to 2Gy IR. Seven days later the cells were fixed and stained with mythelene blue and the clonogenic survival was calculated. Results from 3 independent experiments were averaged and presented as the mean and SE (* = P<0.05 compared to control and # = P<0.05 compared to S2+ alone).
Figure 5.
A proposed model of SESN2-mediated AMPK regulation in response to IR in MCF7 cells.
In response to genotoxic stress (IR), SESN2 is enhanced and leads to the formation of an active LKB1/AMPKα1β1γ1 complex. SESN2 may stabilize the AMPK complex, or transcriptionally regulate AMPK and enhance its expression. Both SESN2 and AMPK may then coordinate mTOR suppression, which translates into enhanced IR-induced cancer cell killing.