Fig 2: p53 stimulates the dissociation-induced ferroptosis in hESCs.A, schematic figure showing p53-mediated activation of ferroptosis. B, p53 and SLC7A11 mRNA expression was determined by qRT–PCR in dissociated hESCs and colony culture. C, Western blot analysis of p53 expression in dissociated hESCs time-dependently. D, densitometric analysis of p53 protein levels; immunoblotting was performed 0–4 h after dissociation of hESCs. E, Western blot analysis of p53 expression in hESCs treated with Pft-a (10 µM). Dissociated cells were treated with Pft-a (10 µM) for 2 h and then dissociated into single cells and cultured for 1 h. F, densitometric analysis of p53 protein levels. G, Western blot analysis of xCT and GPX4 expression in hESCs treated with Pft-a (10 µM). H, densitometric analysis of xCT and GPX4 protein levels. I, mRNA expression of p53 was assessed by qRT–PCR in the presence of DFO (1 µM) or fer-1 (20 µM) along with Y-27632 in dissociated hESCs. GAPDH was used as a loading control. J, Western blot analysis of p53 expression in hESCs treated with DFO (1 µM) or fer-1 (20 µM) in combination with Y-27632. K, densitometric analysis of p53 protein levels; immunoblotting was performed 4 h after dissociation of hESCs. ß-actin was used as an internal control. All the experiments were done 4 h after the dissociation of hESCs. All data are shown as mean ± SEM (n = 3). *p < 0.05, ***p < 0.001, and ****p < 0.0001 compared with the untreated group and, ####p < 0.0001 in comparison to colonies. ††††p < 0.0001 compared with 0 h. $$p < 0.01 compared with 1 h. Fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; hESC, human embryonic stem cell; Pft-a, pifithrin a; qRT–PCR, quantitative RT–PCR; xCT, the cystine–glutamate antiporter.

Fig 3: Rapid cancer modeling with cRNP-edited freshly isolated prostate epithelial cells. (A) Workflow of prostate cancer modeling by cRNP editing of freshly isolated prostate epithelial cells followed by orthotopic transplantation. (B) Western blot showing codisruption of Pten and p53 proteins in freshly isolated prostate epithelial cells. Cells from indicated lineage origin were analyzed 7 d post-cRNP treatment. NT, nontargeting sgRNA control. PP, sgPten+Trp53. (C) Images showing prostate tumor development from mice engrafted with cRNP-edited luminal or basal cells. The dorsal prostate lobes of NSG mice were transplanted with sgPten+Trp53–cRNP-treated luminal or basal cells and harvested at indicated weeks (w) posttransplantation. The urogenital systems or dorsal lobes at harvest are displayed, with an H&E staining showing tumor histology. Dashed circles highlight the prostate tumor regions. (Scale bar, 200 µm.) (D) Histological analysis confirming the engraftment of edited cells in host prostates. The 17-wk endpoint prostate tissues carrying the sgPten+Trp53–cRNP-treated orthografts from C were stained as indicated, with high power magnifications shown in Inset. The lineage origins of the grafted cells are as indicated. A normal gland from the host dorsal lobe is shown in Top. Arrows highlight a gland with a pAkt-positive and p53-negative staining, indicative of Pten Trp53 double knockout. Asterisk, a pAkt/p53 double positive gland, indicative of Pten single knockout. (Scale bar, 200 µm.)

Fig 4: Cytoplasmic RNASE4 binds to and inhibits p53 in human senescent fibroblasts.a Experimental approach and percentage survival of IR-senescent IMR-90 cells 6 days after knockdown of the indicated genes as assessed by MTS assay. Comparisons were made to senescent IMR-90 cells of same cultures infected with Scr-shRNA (100%). b as in a but comparisons were made to senescent IMR-90 cells of same cultures infected with Scr-shRNA or Scr- and p53-shRNA (100%). c Representative images and quantification of proximity ligation assay (PLA) to assess spatial interaction of p53 and Myc-Flag-tagged RNASE4 in indicated irradiated IMR-90 cells. Violin blot shows distribution of PLA signals per cell per IMR-90 replicate. One experiment was performed. Scale bars, 10 µm. Data represent means ± SEM. n = 4 a or n = 3 b independent experiments with each n being an average of 3 technical IMR-90 replicates, n = 3 technical IMR-90 replicates from 1 experiment in c. Statistics: one-way ANOVA with Sidak’s correction in a, b, two-tailed, unpaired t-tests in c. Source data are provided as a Source Data file.

Fig 5: NADH inhibition of SIRT1 activity.a, Lineweaver-Burk transformation of data from SAMDI-MS. b, Relative NADH quantified by HPLC following supplementation with pyruvate or lactate compared to controls (n = 3). c, Densitometric quantification of western blots from liver of 4–6 mo old male mice in indicated genotypes at ZT4 for p53-(K379)Ac (left) and FOXO1-(K242, K245, K262)Ac (right) relative to control TRF-Reg (from Fig. 3d) (n = 3). d, FOXO1 ChIP-seq from liver collected during the daytime comparing effect of FOXO1 binding in TRF-CR (x-axis) with the effect of hepatic SIRT1 ablation (L-Sirt1-/-) (y-axis). Each point indicates a FOXO1 peak in control liver. Peaks that have absolute log2(fold change) >0.5 for both comparisons are colored blue or black and counted by quadrant (n = 2). e, Densitometric quantification of western blots as in (c) relative to null-transduced TRF-Reg mice (from Fig. 3e) (Ac-p53: n = 4 for TRF-CR, LbNOX. n = 6 for all other conditions; Ac-FOXO1: n = 2 for TRF-CR, LbNOX. n = 3 for all other conditions). f, FOXO1 ChIP-seq from liver during the daytime comparing effect of TRF-CR (x-axis) (n = 3) with effect of (f) LbNOX in TRF-CR mice (y-axis) (n = 2) with coloring and counting as in panel d. (n = 2–3). g, Western blotting for Ac-FOXO1 during the nighttime (ZT16) in null- and LbNOX-transduced mice on TRF-Reg and TRF-CR (n.s. non-specific) and densitometric quantification (n = 3). h, BMAL1 ChIP-seq from liver during the daytime comparing the effect of TRF-CR and L-Sirt1-/- as in panel d (n = 3). Uncropped Western blot scans labelled with molecular weight markers are presented in the Source Data Files. i, RNA-seq reads mapping to the exon of Sirt1 that is flanked by LoxP sites (Ex4) relative to exon 9 (Ex9) of Sirt1, and RNA-seq reads per 10 million sequenced reads (RPM) that align to iCre and LbNOX in null- or LbNOX-transduced TRF-CR mice co-transduced with iCre (n = 6). Null-expressing mice on TRF-CR (blue) (n = 5) are shown as reference. j, Quadrant plot comparing transcriptional responses to TRF-CR in null-transduced mice (x-axis) (n = 6) and LbNOX-expression in TRF-CR, L-Sirt1-/- mice (y-axis) (n = 6). Each point indicates a gene that is DE by TRF-CR in null-transduced mice (930 genes). Percentages of genes within each quadrant are shown. k, Average 48-hour fasting body temperature in 4–6 mo old female liver-specific Sirt1-/- mice (n = 5). Data are presented as mean values ± SEM. Statistics were performed with unpaired, two-tailed student’s t test except as otherwise noted in the figure. *p < 0.05. Source data