Extended Data Fig. 5: NUPR1–lipocalin-2 axis reprograms iron metabolism in AT2 cells and LUAD cells.

(a) Measurement of labile iron by Biotracker FerroFarRed dye in KP-RIK LUAD cells following ex vivo transformation of AT2 cells by lentiviral PGK-Cre with or without stimulation with 0.1 µM 2,5-dihydroxybenzoic acid (2,5-DHBA) (n = 3 biological replicates). (b) Quantification of tumor spheres in (a, n = 3 biological replicates). (c) Normalized total cellular iron content measured by ICP-MS in LUAD cells derived from the ex vivo transformation experiment shown in Extended Data Fig. 4i (n = 5, 10, 4, 6, 5, 10, 4, and 6 biological replicates per condition). (d) Normalized labile iron levels measured by Biotracker FerroFarRed dye in LUAD cells derived from the ex vivo transformation experiment shown in Extended Data Fig. 4i (n = 9, 15, 3, 6, 8, 15, 3, and 7 biological replicates). Note that same data in groups without DFO treatment is shown in Fig. 2g. (e-f) Measurement of cellular iron in primary AT2 cells by ICP-MS (total iron, e, n = 4 biological replicates) and staining of BioTracker FerroFarRed dye (labile iron, f, n = 8 biological replicates). (g) Nupr1 mRNA in cultured KP LUAD cells transduced with lentiviral control shRNA (shRenilla) or shRNAs targeting Nupr1 (n = 3 technical replicates from one representative experiment, repeated twice). (h) Alveolar organoid formation by aged vs. young primary mouse AT2 cells stimulated with 50 µg ml⁻¹ iron-loaded mouse transferrin or vehicle control (n = 4, 4, 4, and 3 biological replicates). (i) Alveolar organoid formation by aged vs. young primary AT2 cells in the presence of vehicle control, ZZW-115 (1 µM), or ZZW-115 + 2 µM DFO (n = 4, 4, 4, 4, 4 and 3 biological replicates, left to right). Representative images of alveolar organoids are shown on the right. Scale bar: 100 µm. (j) Violin plots showing expression of Lcn2 in young and aged AT2 cells and LUAD cell states. Two-sided Wilcoxon rank-sum tests on single-cell gene expression vectors were performed for significance testing. (k-m) Lcn2 mRNA level in transformed AT2 cells from the experiment in Extended Data Fig. 4e (k, n = 3 technical replicates), Extended Data Fig. 4f (l, n = 3 technical replicates), and normal alveolar organoids treated with or without NUPR1 inhibitor ZZW-115 (1 µM; m, n = 3 technical replicates). (n) Expression of Nupr1 and Lcn2 in AT2 cells expressing shRNAs targeting Nupr1 or Renilla control in vivo. Gene expression was assessed by single-cell mRNA sequencing of GFP⁺ AT2 cells isolated from the experiment in Fig. 2i. Differential expression was assessed using two-tailed quasi-likelihood F-tests. **, p < 0.01; ***, p < 0.001. Numerical p value is shown in Supplementary Table 18. Y: young, A: aged. Mean with SD is shown in (a-i) and (k-m). Two-tailed Student’s t-test was used in (a), (d) and (f). One-way ANOVA was used in (b-c), (g-i) and (k-m). Two-tailed Mann-Whitney test was used in (e).

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