Extended Data Fig. 4: Nupr1 differentially regulates lung cancer growth in aged vs young mice.

(a) Plots of gene set enrichment analysis (GSEA) showing the enrichment of gene sets linked to iron metabolism in age-related signatures introduced in Extended Data Fig. 3i and Supplementary Table 2 ordered according to fold change. Red indicates enrichment in aged, blue enrichment in young. (b) Violin plots of showing the expression of Nupr1 in young and aged AT2 cells and LUAD cell states. Two-tailed Wilcoxon rank-sum tests on single-cell gene expression were performed for statistical significance. (c-d) Quantification of Nupr1 mRNA level (red dots) by in situ hybridization in AT2 cells (c, n = 1126 and 425 AT2 cells from young and aged animals, respectively) and LUAD cells at 12 weeks post-tumor initiation (d, n = 32 and 12 tumors from young and aged tumor-bearing mice at 12 weeks post-tumor initiation, respectively). Surfactant protein-C (Sftpc) mRNA (green) was used to mark AT2 and cancer cells. Scale bar: 20 µm. (e) Ex vivo transformation of AT2 cells isolated from aged and young KP-Cas9 mice with the indicated lentiviral vectors (Fig. 2c) delivering Cre + control sgRNA or two independent sgRNAs targeting Nupr1 (n = 4 biological replicates). Representative images of transformed tumor spheres are shown on the right. Scale bar: 100 µm. (f) Ex vivo transformation of AT2 cells isolated from aged and young KP-RIK mice, with or without treatment with the NUPR1 inhibitor ZZW-115 (2 µM) (n = 3 biological replicates). (g-h) Quantification of tumor size (g, n = 87, 40, 146, and 121 tumors for young-sgControl, young-sgNupr1, aged-sgControl, and aged-sgNupr1, respectively) and proportion of proliferating (Ki67⁺) tumor cells per total tumor cells (h, n = 71, 133, 40, and 93 tumors for young-sgControl, young-sgNupr1, aged-sgControl, and aged-sgNupr1) in the experiment described in Fig. 2c. (i) Ex vivo transformation of AT2 cells isolated from aged and young KP-Cas9 mice with the indicated lentiviral vectors (Fig. 2c) delivering Cre + control sgRNA or two independent sgRNAs targeting Nupr1, with (grey shading) or without DFO treatment (n = 6 biological replicates except n = 5 in the first and third group). (j) Ex vivo transformation of AT2 cells isolated from aged and young KP-RIK mice by lentiviral PGK-Cre with or without treatment with ZZW-115 (2 µM) and deferoxamine (DFO, 2 µM) (n = 4 biological replicates). (k) Ex vivo transformation of AT2 cells isolated from aged and young KP mice and stimulated with transferrin (50 µg ml⁻¹; n = 3, 3, 4, 4 biological replicates, left to right). (l) Ex vivo transformation of AT2 cells isolated from aged and young KP-RIK mice by lentiviral PGK-Cre with or without stimulation with 50 µM ferric ammonium citrate (FAC) (n = 5 biological replicates). (m) Ex vivo transformation of AT2 cells isolated from aged and young KP-Cas9 mice using lentiviral vectors delivering Cre recombinase and two sgRNAs targeting Nupr1 or a control sgRNA with or without transferrin supplementation (50 µg ml⁻¹; n = 4 biological replicates). Mean with SD is shown in (e-f) and (i-m). Mean with SEM is shown in (g). Median and 25^th and 75^th percentiles are shown by dashed lines in (c-d) and (h). Two-tailed Student’s t test was used in (c-d). One-way ANOVA was used in (e-m).

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