Fig 2: Lcn2 genetic deficiency has minor effects in bleomycin (BLM)-induced pulmonary inflammation and fibrosis. (A) Lcn2 mRNA expression was interrogated with Q-RT-PCR; Values were normalized over the expression of the housekeeping gene B2m and presented as fold change over control; (B) Representative Western blot of Lcn2 expression (red) in lungs from WT and KO mice treated with BLM confirming the global depletion of Lcn2 in KO mice. (C) Weight loss post-BLM administration. (D) Total protein concentration in BALFs, as determined with the Bradford assay. (E) Inflammatory cell numbers in BALFs, as counted with a hemacytometer. (F) Soluble collagen levels in the BALFs as detected with the Direct Red assay. (G) Representative H&E-stained lung sections (×10). (H) Ashcroft scoring of disease severity. (I, J) Indicated respiratory functions were measured with FlexiVent; statistical significance was assessed with one-way ANOVA; */**/**** denotes p < 0.05/0.01/0.0001.

Fig 3: Increased LCN2 mRNA expression in lung epithelial cells of IPF patients, negatively correlating with their respiratory functions. (A) Mean differential LCN2 mRNA expression in IPF patients' cohorts/datasets, detailed in Supplementary Table S1. (B) Volcano plots of differentially expressed genes (FC > 1.2; FDR adjusted p < 0.05) in the three largest transcriptomics datasets of (A) interrogating the expression of 115/44, 28/15, 84/75 IPF patients and controls, respectively (Supplementary Table S1). (C) Spearman's correlation plots of LCN2 expression with spirometry measurements from the GSE47460_GPL14550 cohort (***p < 0.01). (D) Dimensionality reduction plot localizing LCN2 expression in pulmonary epithelial cells originating from 4 IPF patients and 6 controls (26). (E) Dot plot of the same dataset depicting cell type-specific LCN2 expression. The Wilcoxon rank-sum test comparing each cell type with the rest validated LCN2 as a marker gene of the cell types marked in red font (FC > 1.2; Bonferroni adjusted p < 0.05). (F) Per cell type differential expression analysis between cells of different phenotype (IPF vs. control origin) using the Wilcoxon rank-sum test (*FC ≥ 1.2; Bonferroni-corrected p < 0.05; *upregulated in IPF; *downregulated in IPF).

Fig 4: LCN2 BALF levels of IPF patients negatively correlate with their respiratory functions. (A–C) Spearman's correlation plots of LCN2 levels, as measured using a commercially available ELISA, with the ratio of forced expiratory volume (FEV)/forced vital capacity (FVC) (A), with a transfer capacity of the lung for the uptake of carbon monoxide (TLCO) (B) and with carbon monoxide transfer coefficient (KCO) (C). Statistical significance was assessed with Spearman's r = −0.47, −0.42 as indicated; * denotes p < 0.05.

Fig 5: Increased Lcn2 expression in mouse lungs post-BLM-induced pulmonary fibrosis. (A) Mean differential Lcn2 mRNA expression in different transcriptomics BLM datasets (Supplementary Table S1). (B) Volcano plots of the three major datasets of (A). (C) Feature plot showing Lcn2 expression in the mouse lung (27). (D) Dot plot revealing the cell type expression pattern of Lcn2 (in decreasing order of importance). The Wilcoxon rank-sum test comparing each cell type with the rest validated Lcn2 as a marker gene of the cells types marked in red font (FC > 1.2; Bonferroni adjusted p < 0.05). (E) Separate examination of the control (PBS) and fibrotic (BLM) cells further supports the epithelial origin of Lcn2. (F) Per timepoint examination of cell population markers. (G) Bar plot depicting the changes in the mouse lung major cell populations, as defined by scRNAseq analysis clustering and cell typing, across timepoints of BLM administration.