Fig. 1: Deletion of let-7afd in AT2 cells promotes spontaneous lung injury and AT2 transitional cells acutely.

a Quantitative measurement of Sp02 from control, let-7bc2^AT2, let-7afd^AT2 mice (n = 5 per group) following iTAM. Data are mean ± s.d. *let-7bc2^AT2 vs control, ^#let-7afd^AT2 vs control, ^§let-7bc2^AT2 vs let-7afd^AT2, P values were obtained by one-way ANOVA with Tukey’s multiple comparison test. Not significant (NS). Representative lung dissections (b) and H&E-stained lung sections (c) from indicated mice 6-days after iTAM. Scale bars: 5 mm (b); 2 mm upper panel; 50 μm lower panel (c). Arrowheads indicate leukocytes. d GSEA plot shows significant induction of cycling epithelial genes in let-7afd^−/− Sftpc-tdT⁺ AT2 cells compared to controls (n = 3 samples per group). e RNA-seq derived heatmap shows differentially expressed AT2 transition markers in whole lungs or Sftpc-tdT⁺ AT2 cells of let-7afd^AT2 mice compared to controls after 6 days of iTAM. Adjusted p values were derived by two-sided Wald test and BH correction for multiple testing with BaseSpace workflow. f Transcript expression in Sftpc-tdT⁺ AT2 cells after 6 days of iTAM was examined by RNA-seq (n = 3 samples per group). Data are mean ± s.e.m. Adjusted p values vs control were calculated as noted in panel (e). Representative immunostaining (g) and quantification (h) of KI67⁺SFTPC⁺Sftpc-tdT⁺ cells in lungs of let-7afd^AT2 vs control mice 14-days after iTAM. KI67 (gray), SFTPC (green), Sftpc-tdT (red), DAPI (blue). Arrows indicate KI67⁺SFTPC⁺Sftpc-tdT⁺ cells (scale bar 25 μm). (n = 3 mice per group). i Schematic representation to establish AT2 organoid cultures in mice. Created in BioRender. King, K. (2025) https://BioRender.com/x04u478. j Brightfield images show let-7afd^−/− vs control AT2 organoids cultured in AMM. Scale bars 1 mm. k Quantification of CFE and spheroid diameters of organoids in panel (j). (n = 5 per group). Each dot represents one mouse. l GO BP enrichment analysis were calculated from significantly upregulated genes on RNA-seq from let-7afd^−/− compared to control Sftpc-tdT⁺ AT2 cells (n = 3 samples per group) or organoid cultures (n = 2 mice per group). Adjusted p values were determined with a one-sided hypergeometric test with FDR correction with ShinyGo⁵⁸. h, k Data are mean ± s.e.m. P values by unpaired two-tailed Student’s t test. a–c, g, h, j, k Representative of three independent experiments. Source data are provided as a source data file.