Fig 2: Inhibition of Gls1 alters multiple signaling pathways in LSCs and synergizes with PMF in suppressing LSCs. (A–C) GSEA showed enrichment of gene sets with decreased expression including “HALLMARK OXIDATIVE PHOSPHORYLATION”, “NOTCH SIGNALING PATHWAY”, “EWSR1 FLI1 FUSION UP” and (D) increased expression for “HOXA9 DN.V1 UP” in GFP+ LSK cells post BPTES treatment. (E–F) The expression of gene in KEGG pathways were enriched and significantly altered (change fold=2, P < 0.05) post PMF and BPTES treatment. The Venn diagrams demonstrate that the expression of gene in the listed KEGG pathways were decreased post PMF treatment but were increased post BPTES treatment, as well as the expression of genes involved in the listed pathways were decreased after BPTES treatment, but increased after PMF treatment. (G) Left panel: the relative mRNA level of c-Myc, E2f1, Ezh2, Alox5 and Alox15 in RNA-seq data; Right panel: CML mice were induced by BCR-ABL(T315I), on day 14 post BMT, and recipients were treated with Placebo or BPTES (10 mg/kg) for 24 h, then LSCs were sorted with FACS (n = 3, each group). The mRNA levels of BCR-ABL, c-Myc, E2f1, Ezh2, Alox5 and Alox15 in LSCs were examined with RT-PCR. (H) LAMA84 cells were cultured in presence of the indicated concentration of free PM and BPTES for 24 h, then cells were harvested for western blotting analysis with anti BCR-ABL, c-MYC, EZH2, ALOX5, ALOX15, HOXA9, HSP90, P21 and P27 antibodies. GAPDH served as the total protein loading control. (I) A schematic diagram for the regulatory mechanisms of synergistic effects of PM and BPTES. Results were represented with the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered as significant difference.

Fig 3: Ferroptosis of lung alveolar epithelial cells in COPD induced by inflammatory macrophages.a Heat map of normalized enrichment score (NES) following GSEA of cell death pathway gene lists on array data from lung tissue of COPD patients (GSE47460-GPL14550; 145 COPD patients vs 91 healthy controls). b Expression of ferroptotic genes in the lungs of COPD patients relative to healthy controls taken from GSE47460-GPL14550, relative fold change and P value calculated using the GEO2R interactive web tool running limma R. c Western blot analysis of ACSL4 expression in lung core biopsies from healthy (n = 3) and COPD patients (n = 3), quantification relative to vinculin shown for individual patients. d Representative images of immunohistochemical analysis for ACSL4 (brown signal indicated by arrowhead, hematoxylin counterstained, scale bar 25 µm) in lung sections from COPD patients (n = 4) and healthy controls (n = 4). e Quantification of alveolar epithelial cells positive for ACSL4. f Heat map of NES following GSEA of cell death pathway gene lists on array data from mice exposed to 4 m chronic cigarette smoke (CS, n = 3) v filtered air (FA, n = 3). g Expression of ferroptotic genes in the lungs of mice exposed to 4 m chronic CS (n = 3) relative to FA (n = 3) taken from array data used in (f), relative fold change and P value calculated using the GEO2R interactive web tool running limma R. h, i Cells from whole lung suspensions of mice exposed to FA (n = 3) or CS for 4 m (n = 5), were analysed by scRNA-Seq (Drop-Seq). h UMAP of scRNA-Seq profiles (dots) colored by cell type. i UMAP plots showing expression of ACSL4 in scRNA-Seq profiles. j Representative images of immunohistochemical analysis for ACSL4 (red signal indicated by arrowheads, hematoxylin counterstained, scale bar 25 µm) in lung sections from WT and Prmt7+/- mice exposed to FA or CS for 4 months (n = 4–5 per group). k Quantification of alveolar epithelial cells positive for ACSL4 from (j) (n = 5 for WT FA, Prmt7+/- FA, Prmt7+/- CS and n = 4 for WT CS). l, m Mouse AT2 cells (MLE12) were treated with RSL3 (250 nM), or conditioned medium from control macrophages (M?, RAW264.7 cells) or those polarized to a pro-inflammatory (IFN? + LPS) or anti-inflammatory (IL4) phenotype for 48 h, representative data shown from a single experiment repeated twice. l Representative image showing PI uptake at 24 h (Scale bar 50 µm). m Number of dead AT2 cells at the time indicated, data shown from a single experiment repeated twice. n BODIPY oxidation in AT2 cells (MLE12) at 6 h after treatment with RSL3 (250 nM, n = 3) or conditioned medium from control macrophages (n = 2) or those polarized to a pro-inflammatory (n = 6) or anti-inflammatory (n = 3) phenotype. o Number of dead AT2 cells (MLE12) by PI uptake at the time indicated following treatment with RSL3 (250 nM), or conditioned medium from macrophages (M?, RAW264.7 cells) polarized to pro-inflammatory (IFN? + LPS) plus 20 µM zVAD or 10 µM necrostatin-1s (Nec1-s), representative data shown from a single experiment repeated twice. p Western blot analysis of ACSL4 in AT2 cells (MLE12) treated for 24 h with conditioned medium from control macrophages (n = 11) or those polarized to a pro-inflammatory (n = 10) or anti-inflammatory (n = 5) phenotype, normalized to ß-actin and shown relative to control macrophages. q Western blot analysis of ALOX5 at 72 h in control macrophages (n = 11) or those polarized to a pro-inflammatory (n = 11) or anti-inflammatory (n = 11) phenotype, normalized to ß-actin and shown relative to control macrophages. r Western blot analysis of ACSL4 in AT2 cells (MLE12) treated for 24 h with conditioned medium from control macrophages or those polarized to a pro-inflammatory phenotype that were treated with siRNA against Alox5, normalized to ß-actin and shown relative to control macrophages, from individual experiments (n = 3). Data shown mean ± SD, P values shown in charts determined by unpaired two-tailed Student’s t-test (c, n), two-tailed Mann–Whitney test (e), one-way ANOVA Bonferroni’s multiple comparisons test (k, p, q). Source data are provided as a Source Data file.

Fig 4: PRMT7 expression is increased in COPD lungs and localized to macrophages.a Heat map of the most significantly enriched gene lists in the lungs of COPD patients following gene set enrichment analysis (GSEA) of the GO molecular function set on publicly available array data from lung tissue (GSE76925) of healthy smokers (n = 40) v COPD patients (n = 111). Nominal P value generated by the GSEA software of the enrichment score relative to the null distribution92 shown. b Expression of genes involved in N-methyltransferase activity in COPD patients relative to healthy smokers taken from GSE76925, relative fold change, and P value calculated using the GEO2R interactive web tool running limma R. PRMT genes highlighted in red. c, d Expression of PRMT7, in the lungs of healthy smokers and COPD patients, individuals are shown, expression relative to healthy smokers, taken from GSE76925 (n = 40 smokers and n = 111 COPD patients) (c) and GSE27597 (n = 16 smokers and n = 48 COPD patients) (d). e qPCR analysis of PRMT7 and TNF expression in human lung core biopsies from healthy controls (n = 8) and COPD patients (n = 18) relative to controls. f Western blot analysis of PRMT7 expression in lung core biopsies from healthy (n = 17) and COPD patients (n = 23), normalized to ß-actin and shown relative to controls. g Representative images of immunofluorescence analysis for PRMT7 (Red) and the macrophage marker Galectin 3 (Green) in sections from core biopsies of healthy and COPD human lung (n = 4, scale bar 20 µm) and sections from filtered air (FA) and cigarette smoke (CS) mouse lung (n = 5, scale bar 25 µm). h Quantification of double-positive cells from (g). i, j mRNA expression levels of PRMT7 determined by qPCR in human monocytes isolated from blood of non-smokers (n = 7) vs smokers (n = 10) (i) and circulating monocytes (n = 4 for FA and CS) (i) and alveolar macrophages (n = 4 for FA and n = 3 for CS) (j) from B6 mice exposed to FA or CS for 3 days, relative to controls. k–m scRNA-Seq (Drop-Seq) analysis on the lungs of mice following exposure to FA (n = 3) and CS for 2 (n = 5) and 4 months (n = 5). AM alveolar macrophages, CS-ind MØ CS-induced macrophages, IM interstitial macrophages, cMono classical monocytes. k Dot blot depicting Prmt7 expression (log-transformed, normalized UMI counts) and percentage of cells positive for Prmt7 in the myeloid cell compartment. l RNA velocity analysis of the myeloid compartment. (1) AM; (2) CS-ind MØ; (3) Lyve1 + /Cd163 + IM; (4) Lyve1-/Cd163- IM; (5) Prg4 + IM; (6) Ly6c2 + cMono; (7) Ly6c2- non-cMono; (8) Cd103 + /Clec9a + cDC; (9) Cd209 + /Cd11b + cDC; (10) Fscn1 + DC. m Fate probability mapping towards the CS-ind MØ population utilizing CellRank. n Heat map of gene expressions of PRMT6/7, RAP1A, and ALOX5 in monocyte-like macrophages obtained from scRNA-Seq data of bronchoalveolar lavage from COPD patients (n = 9) and healthy controls (n = 6). Mean gene expression per donor is shown as a z-transformed value (across all donors). Data shown mean ± SD and individual patients or mice (c–f and h–j), P values shown in charts determined by two-tailed Mann–Whitney test (c–f), unpaired two-tailed Student’s t-test (h–j). Source data are provided as a Source Data file.

Fig 5: The free PM shows potent suppression activity on Ph +cell lines. (A) LAMA84 cells were cultured in presence of 20 nM free PM for indicated time, then cells were harvested for westren blotting analysis using Ac-TUBULIN,TUBULIN, Ac-H3, and H3 antibodies. GAPDH served total protein loading control. (B) LAMA84 and K562 cells were transfected with the mCherry-luciferase-puromycin virus to generate stable cell lines, and luciferase-tagged cells were cultured in presence of DMSO or the indicated concentration of free PM. Luminescence activity was measured every 12 h. Relative cell vitality was normalized with cells treated with DMSO. (C) BCR-ABL(T315I) or MIG transducted BaF3 cells were treated with indicated concentration of free PM. Cell apoptosis was measured with Annexin-V/PI staining and analyzed with FACS. (D) LAMA84 and K562 cells were cultured in presence of DMSO or free PM at the indicated concentrations for 24 h, the harvested cells were digested with RNAase and stained with PI for cell cycle analysis. (E) LAMA84 and K562 cells were cultured with DMSO or the free PM at the indicated concentrations for 36 h. Cell apoptosis was measured with Annexin-V/PI staining and analyzed with FACS. (F) LAMA84 cells were treated with 20 nM free PM for the indicated time, and western blotting analysis were performed to detect endogenous and exogenous apoptotic proteins including CASPASE9, CASPASE8, CASPASE3, PARP and their associate proteins P53, BCL-XL and BAD; (G) and proteins involved in cell proliferation pathway including c-RAF, p-c-RAF, MEK, p-MEK, ERK, p-ERK, AKT, p-AKT; and (H) BCR-ABL, c-MYC, ALOX5, ALOX15, ß-CATENIN, EZH2, p-STAT5, STAT5, p-FAK, and FAK. GAPDH served as total protein loading control.