Fig. 1: Rewiring of cellular metabolism accompanies airway epithelial cell differentiation.

a FACS gating strategy of murine tracheal epithelial cell cultures (mTEC) collected at ALI day 10 (CD49f⁺NGFR⁺ basal cells; CD49f⁻NGFR⁻ differentiated cells). b Ingenuity Pathway Analysis of differentially expressed genes obtained from the RNA sequencing of basal and differentiated cells (p adj <0.01), to identify differentially regulated pathways. The indicated Log₁₀(p-values) were calculated using the Fisher’s Exact test. The heatmap showing the expression of transcripts related to the ‘fatty acid metabolism’ pathway was generated using the Broad Institute ‘Morpheus’ software. c Gene set enrichment analysis (GSEA) of the ‘metabolism of lipids and lipoproteins’ pathway. NES: normalized enrichment score; FDR: false discovery rate. d Flow cytometry analysis of CD36 expression on the surface of basal and differentiated cells at ALI day 10. Data show geometric mean +/− SD. Two-tailed unpaired Student’s t test; n = 3 biologically independent samples representative of 3 independent experiments. MFI: mean fluorescence intensity. e Flow cytometry analysis of mTEC cultures (ALI day 10) exposed to either green-fluorescent fatty acid bodipy-C16, fluorescent glucose analogue 2-NBDG, TMRM or mitotracker orange for 30 min at 37 °C. Cells were then stained to define the basal and differentiated subsets. Mean +/− S.D.; p values were calculated using unpaired, two-tailed t test; n = 3 biologically independent samples from 3 independent experiments. f, g Volcano plots showing the distribution of intracellular lipid species identified and quantified by mass spectrometry analysis of FACS-sorted basal and differentiated cells. Plot in f shows data normalized to cell number; plot in g shows data upon quantile normalization, to highlight distribution of lipid species between cell types. Dashed black lines indicate the fold change filter of FC > 2 and FC < −2 and the p-value filter of p < 0.05, based on two-tailed, unpaired t tests, with unequal variance, FDR correction. h Box plots show the relative abundance of lipid species in FACS isolated basal and differentiated cells. Lines indicate median values, whiskers indicate minimum and maximum values, and boxes are limited by the 25th and 75th percentile. Plot shows data of five independent samples.