(A) Luciferase assay reporter gene constructs containing the putative p53 recognition sequences within the GAMT promoter were co-transfected into Saos2 cells with either wild-type p53 or mutant p53 (V143A) expression construct, as well as a control pcDNA3.1 empty vector. The same reporter constructs were also transfected into U2OS cells followed by ETO treatment. The reporter plasmids used were the following: empty pGL4 vector, the full-length (P1) promoter in pGL4, and three reporter constructs with p53-binding site deletions or specific mutations (P2, P3, and M1); the p21 promoter was used as a control. The upper panel shows putative p53-recognition sequences with the GAMT promoter and the p53-consensus binding sequences (BS). Error bars represent ± S.D. (n=3).
(B) For EMSA, nuclear extracts (N.E.) from U2OS cells infected with Ad-p53 were incubated with radiolabeled wt-GAMT BS1 oligonucleotides in the presence of cold specific or non-specific mutant competitor and, where indicated, pAb421 antibody. The migrating positions of bound tetramer and super shifted antibody-p53-DNA complex are indicated as arrows.
(C) U2OS cells were treated with ETO (25 μM) for 24 hours. Saos2 cells were infected with Ad-p53 or Ad-GFP for 24 hours (lower panel). ChIP assay was then performed with either a p53 (DO-1 Ab) antibody or an HA-tag antibody (negative control). The % input of co-precipitating DNAs were calculated by qPCR and presented as mean ± S.E.M (n=3). The GAMT p53 BS1 amplification product from ETO treated U2OS cells is shown in the agarose gel and p21 was used as a positive control.

(A) EJ-p53 cells were transfected with shRNA against GAMT (shGAMT) or luciferase as a control (shControl), and p53 and its target Mdm2 were induced after tetracycline (tet) removal. At 48 hours, the levels of guanidino compounds creatine (Cr), creatinine (Crn) and guanidinoacetate (GAA) were measured in normalized cell lysates. The ratio between GAA and creatine + creatinine was calculated in p53-induced cells in comparison to the levels found in control cells (right panel). Error bars represent ± S.D. (n=3).
(B) HCT116 and U2OS cells were transfected with either GAMT shRNA (shGAMT) or control luciferase shRNA (shCont.), and then treated with ETO (25μM). At 24 hours, the levels of guanidino compounds were measured. Error bars represent ± S,D. (n=3).
(C) HCT116 and U2OS cells were treated with cyclocreatine (cCr, 2.5 mM) and then treated with ETO (25μM) for 36 hours. Apoptosis was measured by TUNEL assay. Error bars represent ± S.D. (n=3).
(D) The ratio between the mean of GAMT mRNA levels in wt-mice and the mean of p53−/− counterparts is described for the indicated tissues and is presented as mean ± S.E.M. (left panel, n=3). GAMT protein expression was also measured in liver and pancreas tissues. (E) The value of each guanidino compound in several tissues was determined in p53−/− mice relative to wt-mice, which was set equal to 100%, and shown by mean value ± S.D. (n=3).

(A) GAMT depleted HCT116 p53+/+ cells (pooled #1 and #2) were grown in the absence of glucose for the indicated times (0–2 days). Cell lysates were analyzed by immunoblotting with antibodies against GAMT, PARP (cleaved), caspase-3 (cleaved), caspase-9 (cleaved), and β-actin (loading control).
(B) HCT116 cells were transfected with either GAMT shRNA #1 or #2, or control (luciferase) shRNA, then cultured in the absence or presence of glucose for the indicated times. The percentage of dead cells was determined by trypan blue exclusion analysis, TUNEL assay, and PI staining-FACS analysis.
(C) HCT116 p53−/− cells stably expressing control and GAMT shRNA (#1 and 2) were cultured with or without glucose for 30 or 48 hours. The cells were then harvested for western blot analysis to confirm the knockdown of GAMT expression. Cells were further assessed for cell death/apoptosis by PI staining-FACS analysis and TUNEL assay for the indicated times. Error bars indicate ± S.D. (n=3), with duplicate plates.
(D) HCT116 p53−/− cells transiently transfected with GAMT expression plasmid or pcDNA3.1 vector were cultured with or without glucose for 24 or 48 hours. Cells were assessed for GAMT expression and for cell death/apoptosis by TUNEL assay for the indicated times. Error bars indicate ± S.D. (n=3), with duplicate plates.
(E) Control (luciferase) and GAMT (#1) knockdown HCT116 cells were cultured with or without glucose, harvested at 0, 36 and 48 hour. Fractionated lysates were assessed for the presence of cytochrome c, oxidative complex 1 (OC1, mitochondrial fraction), and IκBα (cytosolic fraction) by immunoblot analysis.

(A) GAMT mRNA and protein were measured after tetracycline (tet) removal in EJ-p53 cells (tet-off). Saos2 cells were infected with adenovirus expressing wt-p53 (Ad-p53) or GFP (Ad-GFP) for 24 hours.
(B) Saos2 and U2OS cells were treated with etoposide (ETO, 25 μM) or its solvent (DMSO) for 12 or 24 hours. In addition, U2OS cells were transfected with either siRNA oligos targeting p53, or control siRNA (luciferase), followed by exposure to ETO for 12 or 24 hours.
(C) Saos2 (p53-null), U2OS (wt-p53), and A549 (wt-p53) cells were treated with H₂O₂ (1 mM), and U2OS cells were exposed to ionizing radiation (IR, 10 Gy) for the indicated time.
(D) MEFs (mouse embryonic fibroblasts) were infected with adenovirus expressing wt-p53 (Ad-p53) or GFP (Ad-GFP). p53+/+ and p53−/− MEF cells were treated with DMSO or ETO (25μM). mRNA levels were normalized to 36B4 expression and presented as mean ± S.E.M (n=3).

(A) U2OS cells were transfected with either GAMT shRNA (shGAMT#1 and #2, respectively) or control luciferase shRNA (shCont.), and then treated with ETO (25μM) for the indicated times. Protein extracts were immunoblotted, and cells were also assessed for apoptosis by TUNEL assay (the middle panel). In the right panel, the percentage of TUNEL positive cells is shown as the mean ± S.E.M. (n=3).
(B) Saos2 cells were transfected with GAMT shRNA or control shRNA, followed by infection with adenovirus expressing GFP (Ad-GFP) or p53 (Ad-p53) for the indicated times. The apoptotic response of Ad-infected Saos2 was assessed by TUNEL assay and PI staining. In all cases, error bars indicate ± S.D. (n=3).

(A) Saos2 (p53-null), U2OS, A549, A172 and IMR90E1A cells were cultured in the absence of glucose for the indicated times, and protein extracts were immunoblotted with antibodies against phospho-AMPKα, p53, phospho-Ser¹⁵p53, GAMT and β-actin.
(B) HCT116 p53+/+ and HCT116 p53−/− cells were cultured in the absence of glucose for the indicated times (0–48 hours), and the levels of p53, p21, GAMT, phospho-AMPKα, and β-actin were examined. HCT116 cells were transfected with either siRNA-oligos targeting p53, or control siRNA oligos, followed by glucose withdrawal for the indicated times. In addition, the levels of guanidino compounds were measured. Error bars indicate ± S.D. (n=3).
(C) The calculated percent increases of GAMT mRNA upon starvation (24 hours) in wt-mice of various tissues and presented as mean ± S.E.M. (n=3) (the top panel). The levels of GAMT proteins in liver and pancreas of wt-mice and p53-null mice under fed or starved condition were determined (the bottom panel).
(D) Guanidino compounds in serum and liver was determined in starved wt-mice relative to fed mice, which was set equal to 100%, and shown by mean value ± S.D. (n=3).

(A) HCT116 cells were grown in complete medium containing glucose or no glucose, with or without FAO inhibitor etomoxir (50 μM), with or without a creatine kinase inhibitor, cyclocreatine (cCr, 2.5 mM and 5 mM, respectively) for 24 hours, and relative ATP levels were measured. Error bars indicate ± S.D. (n=3).
(B) HCT116 cells were transfected with p53 shRNA, GAMT shRNA or control shRNA, and then incubated with or without glucose for 24 hours, followed by fatty acid oxidation measurement.
(C) Saos2 cells were transfected with wt-p53, mutant p53, GAMT, or empty vector pcDNA3.1 as a control, and the rate of fatty acid oxidation was measured. The values were shown by mean ± S.D. (n=3), with duplicate plates.
(D) HCT116 p53+/+ or HCT116 p53−/− cells were transfected with either GAMT or pcDNA3.1 empty vector (control), then maintained in the absence or presence of glucose for 24 hours, followed by fatty acid oxidation measurement.
(E) Fatty acid oxidation was measured in HCT116 cells that were treated with or without creatine (Cr, Sigma) at 5 mM and 10 mM for 24 hours. Cells were treated with AICAR (2 mM) for 2 hours as positive control. Values shown are mean ± SEM (n=6). Cell lysates were subjected to immunoblotting with phospho-AMPKα and phospho-ACC antibodies.
(F) Fatty acid oxidation was measured in liver tissues from fed or starved (24 hours) wt-mice and p53-null mice. Error bars indicate ± S.D. (n=3).
(G) Model for the role of p53→GAMT pathway in creatine biosynthesis and fatty acid oxidation pathway.