Extended Data Fig. 6: Activation of NUPR1–lipocalin-2 axis leads to ferroptosis resistance in aged LUAD cells.
From: Ageing limits stemness and tumorigenesis by reprogramming iron homeostasis
(a) Ex vivo transformation of AT2 cells isolated from aged and young KP mice with lentiviral PGK-Cre + shRNAs targeting the indicated genes (n = 3 biological replicates). Data are shown as fold change compared to shRenilla control. (b) Measurement of labile iron in LUAD cells derived from Fig. 3e; data is shown normalized to control sgRNA. N = 9, 15, 5, 8, 8, 15, 5, and 10 biological replicates from left to right, respectively. Note that data points of sgControl and sgNupr1 without Lcn2 cDNA is the same as in Fig. 2g. (c) Ex vivo transformation of AT2 cells isolated from aged vs. young KP-RIK mice using lentiviral PGK-Cre, with or without 100 ng ml−1 recombinant mouse lipocalin-2 and/or 2 µM ZZW-115 (n = 4, 4, 4, 3, 4 and 4 biological replicates, left to right). (d) Measurement of labile iron by Biotracker FerroFarRed dye in KP LUAD cells derived from ex vivo transformation of aged and young AT2 cells treated with anti-lipocation-2 neutralizing antibody (5 µg ml−1, n = 6 biological replicates). (e) Ex vivo transformation of AT2 cells isolated from aged vs. young KP-Cas9 mice with PGK-Cre and control sgRNA or sgRNAs targeting Lcn2, with or without treatment of anti-lipocation-2 neutralizing antibody (5 µg ml−1, n = 4 biological replicates). (f) Validation of shRNA targeting Gpx4 by quantitative PCR (n = 3 technical replicates per condition from a representative experiment, repeated twice). (g) Ex vivo transformation of aged and young AT2 cells of KP mice with shRNA targeting Gpx4 (n = 6 biological replicates). Data are shown as fold change compared to shRenilla control. (h) Ex vivo transformation of AT2 cells isolated from aged vs. young KP mice with PGK-Cre, with or without NUPR1 inhibitor ZZW-115 (2 µM) and ferroptosis inhibitor liproxstatin-1 (5 µM). N = 4 biological replicates. (i) Ex vivo transformation of AT2 cells isolated from aged vs. young KP mice using lentiviral PGK-Cre, with or without mouse transferrin or ferroptosis inducer RSL-3 (n = 3 biological replicates). Mean with SD is shown in (a-i). Two-tailed Student’s t test was used in (a), (d) and (f-g). One-way ANOVA was used in (b-c), (e) and (h-i).
