Fig 2: Association of p53 with a secondary enhancer site in the p21 intron.(A) Map of the human p21 gene. A sequence alignment of the consensus p53 response element (RE) with the upstream enhancer at position -2.3 kb and the intronic enhancer at position +4.0 kb is shown underneath, and the relative position and transcript structure of two spliced EST clones is indicated above. (B) ChIP profile of p53, and (C) of BRD4 across the p21 gene in the absence or presence of 067. D. Local enrichment of the indicated factors within the transcription unit of p21. ChIP values of cells treated with 10 µM 067 for 4 hours were calculated as fold enrichment over DMSO-treated control samples (dashed line) for each position individually.

Fig 3: DRG2 inhibits SIRT1-mediated deacetylation of p53. (a)–(e) pcDNA-hSIRT1 (800 ng) was cotransfected into WI-38 cells with increasing doses (100, 200, and 400 ng) of DRG2-expressing plasmid or empty control for 18 h before exposure to H2O2 (200 µM, 72 h). (a) Expression of SIRT1, ac-p53 (Ly382), p53, and ß-actin was analyzed by western blotting. (b) Ac-p53 (Ly382) was quantified by densitometry based on immunoblot images. ß-Actin was used as a loading control. (c) SIRT1 deacetylase activity was measured using a SIRT1 fluorescent activity assay. (d) and (e) Representative images of SA-ß-gal staining of WI-38 cells (100× magnification). The percentage of senescent cells was calculated from 3 random regions. (f) (1-2) WI-38 cells were treated with or without H2O2 (200 µM, 72 h). Expression of endogenous DRG2 (red) and SIRT1 (green) was assessed by immunofluorescence staining, along with nuclear counterstaining using DAPI (blue). (3) WI-38 cells were cotransfected to express both pEGFP-DRG2 (400 ng) and pcDNA-hSIRT1 (800 ng) plasmids for 18 h and grown in complete media for 72 h. Expression of DRG2 (green) and SIRT1 (red) was assessed by immunofluorescence staining, along with nuclear counterstaining using DAPI (blue). Scale bar, 10 µm. Data are presented as the mean ± SEM value for each treatment. Similar results were obtained from three independent experiments. *P < 0.05 versus control. †P < 0.05 versus H2O2-treated cells.

Fig 4: Fasting attenuates hypothalamic SIRT1 activity and hypothalamic FGF21-Oxt signalling. a The effects of feeding and fasting on hypothalamic NAD+ levels (n = 12 in the Fed group, n = 13 in the Fasted group). b Photomicrographs depict hypothalamus coronal section from wild-type C57BL/6 mice (fed or fasted state) that received 3rd ICV injection of 0.5 µl of TSA (10 µg/µl). The length of scale bars is 100 µm. The sections were immunostained to identify Ac-FOXO1, Ac-p53 and Ac-NF-?B protein (black or blue) expression. c, d The effects of feeding and fasting on hypothalamic expression levels of Klb (c) (n = 6 in the Fed group, n = 7 in the Fasted group) and Oxt (d) (n = 6 in the Fed group, n = 7 in the Fasted group) in C57BL6/J mice fed ad libitum or fasted for 24 h. e Hypothalamic Oxt mRNA expression levels after IP injection of mFGF21 (1 mg/kg) or water (vehicle) in 24-h–fasted C57BL6/J mice (n = 4 per group). f Schematic diagrams of hypothalamic FGF21–Oxt signalling and SIRT1 in fasting and feeding. (Left) Fasting increased circulating FGF21 levels but suppressed hypothalamic SIRT1 activity, which was associated with the down-regulation of FGF21–KLB–OXT signalling. (Right) During simple sugar feeding, hypothalamic SIRT1 activity was maintained, and FGF21 functioned as a metabolic signal for regulating the simple sugar preference through the FGF21–OXT signalling. Data are shown as box and whisker plots (centre line, median; box limits, upper and lower quartiles; whiskers, the minimum and maximum value of a data set). *p < 0.05. 3V, 3rd ventricle; Ac. acetylated. See also Supplementary Fig. 8

Fig 5: MAGEA2 overexpression is functionally linked to TR. (a) Western blot analysis of lysates (20 µg) from MCF-7-stable clones (C18 and C24) expressing exogenous MAGEA2, plus the VA control line, was probed for MAGEA2, p53, acetylated p53 and p21cip, as indicated. Blots were reprobed for Hsc70 (loading control). (b) MCF-7 (WT and VA) and clones C18 and C24 were split into six-well plates (5 × 105 cells per well) and treated with 10-7M tamoxifen (TAM) 24 h later for 8 days. Triplicate wells were harvested and counted daily. (c) MCF-7 VA and C24 lines were split into 12-well plates (5 × 104 cells per well) and treated with a range of tamoxifen concentrations, as indicated, over 8 days. For statistical analysis, data from all the C24 samples were averaged and compared with the averaged VA data. (d) MCF-7 control (WT and VA) and clones C18 and C24 were plated in triplicate on six-well plates (105 cells per well), grown with or without 10-7M tamoxifen for 144 h, and then assayed for Annexin V binding and PI uptake. The graph shows the percentage of early apoptotic (AnnV+PI-) cells for each line and the condition (see Supplementary Figure S4A for full analysis). (a–d) Error bars indicate the s.e. Student's t-test was used to compare data from the indicated sample with VA control; *P<0.05, **P<0.01. (e) Western blot analysis of lysates (20 µg) from T47DTR cells transiently transfected with siRNA targeting MAGEA2 or non-silencing control (NSC) for 48 h probed for the proteins indicated. (f) T47DTR cells were transfected with NSC or siRNA targeting MAGEA2. After 48 h, transfected cells and WT T47D were split into 24-well plates (3 × 104 cells per well in triplicate) and treated with 10-6M tamoxifen 24 h later for 5 days. Error bars indicate the s.e. Student's t-test was used to compare data from NSC- and siMAGEA2-transfected cells; *P<0.05.