Research Paper Volume 1, Issue 5 pp 490—502

Figure 6. Activation of p53 by metabolic stress; effects of an AMPK inhibitor on p53 phosphorylation. (A) An AMPK inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (B) An AMPK inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (C) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (D) An ATM inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.