Fig 2: PFKL is a physiologically relevant target of TAp73. a Schematic representation of human PFKL genomic structure. The sequences of potential p73 response elements RE1–3 and the corresponding mutant RE3 are shown. b, c Luciferase constructs containing RE1, RE2, and RE3 (b), or RE3 and mutant RE3 (c) were transfected into 293T cells together with Flag-TAp73a or vector control. Renilla vector pRL-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. Data are means ± S.D. (n = 3). d Luciferase reporter constructs containing RE1, RE2, RE3, or RE3mut were transfected into 293T cells together vector control, p53, or TAp73. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data are means ± S.D. (n = 3). Insert shows protein expression. e, f U2OS cells (e), or 293T cells transfected with control vector or Flag-TAp73 (f), were analyzed by ChIP assay using normal mouse IgG and anti-p73 antibody (e), or anti-Flag antibody (f). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. g, h U2OS (g) and HCT116 (h) cells transfected with the indicated siRNAs were analyzed for protein and mRNA expression. Results are representative of three independent experiments. i, j U2OS cells (i), or 293T cells transfected with Flag-p53 or vector control (j), were analyzed by ChIP assay using normal mouse IgG and anti-p53 antibody (i), or anti-Flag antibody (j). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. k p53-/- HCT116 cells stable expressing Tet-inducible p53 were cultured in medium containing [1,2–13C2]glucose and treated with doxycycline to induce p53 expression (Tet-on). p53, p73 and PFKL expressions were determined by Western blot analysis. TIGAR expression was analyzed by qRT-PCR. (means ± S.D., n = 3). Relative glycolytic flux is shown in Supplementary Fig. 4b

Fig 3: CB002 and structural analogs restore the p53 pathway, whereas other xanthines caffeine, pentoxifylline, and theophylline do not.CB002 structural analogs activate p53 reporter gene activity in SW480 cells in a dose-dependent manner (6 hr) (A). Therapeutic indices for CB002-structural analogs were determined in SW480 cells (48 hr) (B). Propidium iodide cell cycle analysis was performed to determine sub-G1 population at 48 hr of treatment with CB002-analogs at 100 µM in SW480 cells. Two-way ANOVA, p<0.05 (C). CB002-analog #4 restores the p53 pathway in SW480 cells, resulting in PARP cleavage independently of p73 (D). Immunofluorescence staining of Cyt-C (green), Tom20 (red) DAPI (blue) in SW480 treated as indicated for 48 hr (E). Noxa protein expression induced by CB002-analogs in DLD-1, SW480, HCT116, and HCT116 p53(R175H) colorectal cancer cells (24 hr) (F). p53-pathway restoring compounds have unique properties compared to other xanthine derivatives in their ability to induce Noxa expression, 24 hr treatment in DLD-1 cells (G). Xanthine derivatives CB002 and its analog induce Noxa expression but not caffeine, pentoxifylline, and theophylline at 24 hr in DLD-1 and SW480 cells (H). ATF3/4 mediate Noxa induction (I). Caffeine (C), Pentoxifylline (P), and Theophylline (T). Figures (A)–(C) were performed as three biological replicates. Experiments from figures (D)–(I) were performed at least twice and a representation of one is shown.

Fig 4: TAp73 regulates the expression of PFKL. a Relative mRNA levels of glycolytic enzymes in TAp73+/+ and TAp73-/- MEFs (clone #1), as analyzed by qRT-PCR. Data are means ± S.D. (n = 3). b, c TAp73+/+ and TAp73-/- MEFs (clone #1 and #2) were analyzed by qRT-PCR for expressions of PFKL, PFKM, and PFKP, and G6PD. Data are means ± S.D. (n = 3). d TAp73+/+ and TAp73-/- MEFs (clone #2) were analyzed by semi-quantitative RT-PCR (top), and Western blot (WB, bottom). e mRNA levels of PFKL, PFKM, and G6PD in ?Np73+/+ and ?Np73-/- MEF cells. Results are representative of three independent experiments. f U2OS cells were transfected with control or p73 siRNA as indicated. mRNA expression was detected by qRT-PCR (means ± S.D., n = 3). g, h U2OS cells were transfected with control or TAp73 siRNA were analyzed by qRT-PCR (g, means ± S.D., n = 3), semi-quantitative RT-PCR (h, top), and Western blot (WB) (h, bottom). i U2OS, HCT116, and H1299 cells transfected with control or TAp73 siRNA were analyzed by Western blot. Results are representative of three independent experiments. j, k HeLa cells transfected with control or TAp73 siRNA were analyzed for mRNA and protein expression. Data are means ± S.D. (n = 3). l U2OS cells stably expressing control plasmid or an siRNA-resistant TAp73 plasmid were transfected with a control or TAp73 siRNA. PFKL expression was analyzed by RT-PCR and Western blotting

Fig 5: Inhibition of aurora kinases induces cellular senescence in vitro independent of p53Hs294T (p53WT), SK-Mel-2 (p53 mutant), SK-Mel-5 (p53WT) and SK-Mel-28 (p53 mutant) cells were treated with 1 µM MLN8237 for 6 or 12 days. After treatment, viable cells were counted and compared to the initial cell number. Data indicate mean values ± SD (n = 3) from one representative of three independent experiments. p-value shown represents difference between treated group and day 0 control group (Student's t-test).Cultures of Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with MLN8237 or vehicle for indicated time points and apoptosis was analysed by FACS analysis for propidium iodide (PI) and Annexin V staining. Data indicate mean values ± SD (n = 3) from triplicate experiments.Hs294T cells were treated with 1 µM MLN8237 or vehicle for 5 days and the change in morphology was captured with an AxioVision microscope.Hs294T cells were treated with 1 µM MLN8237 for 5 days, and senescence was determined by ß-galactosidase staining.Melanoma cell lines with wild-type p53 (Hs294T and SK-Mel-5) or mutated p53 (SK-Mel-2 and SK-Mel-28) were treated with 1 µM or 5 µM MLN8237 for 5 days. After treatment, p53, p63, p73, p21, p16 and GAPDH were analysed by Western blot.Hs294T and SK-Mel-28 cells were treated with 1 µM MLN8237 or vehicle in the presence or absence of the p53 inhibitor pifithrin-a (PTF-a, 5 µM) or DMSO for 5 days. After treatment, ß-galactosidase staining was performed. All experiments were conducted at least three times independently with reproducible results.