Fig. 7: C/EBPα downregulation is required to re-access mutually exclusive Defender and Regenerative states in response to injury.

a Experimental design for AAV Cebpa deletion study (top). Immunostained lungs 14 days post injury (bottom) showing GFP⁺ AT1s (asterisk). Bars, 100 µm. b Quantification of (a) percent GFP⁺ AT1s (n represents GFP⁺ cells sampled per condition in experimental triplicate). Data as mean ± SD (Student’s two-sided t test). c Adult mouse lung 16 days post Bleomycin injury immunostained for ECAD (green), C/EBPα (red), and DAPI (blue). Lower panel depicts C/EBPα expression (fire LUT) in injured versus uninjured sites. Bars, 100 µm. d Quantification of (c) mean C/EBPα intensity of AT2s within uninjured or injured regions. Data are represented as box plots showing median, upper, and lower quartiles, and the whiskers extend to the minima and maxima. p values determined using two-tailed Mann–Whitney U-test (n represents number of distal alveolar buds sampled per condition). e Dot plot of AT2s from published scRNAseq 1X Bleomycin timecourse study (GSE141259) depicting genes of known C/EBPα suppressors (Ddit3, Atf3, Atf4, and Ezh2—upregulation indicated in red) as well as DATP gene score. f Experimental design for stimulating CHOP in vivo. Control or CT020312 treated PN25 lungs immunostained for AT2 lineage (GFP) and MUC1 (white). Bars, 50 µm. g Quantification of (f) showing percent GFP⁺ AT1s in control and treated condition (n represents GFP⁺ cells sampled per condition). Data as mean ± SD (Student’s two-sided t test). h Leiden clustering identifies two Cebpa^fl/fl AT2 clusters: AT2^DLK1+ (green) and AT2^Def (red) which distinct velocity trajectories. i Gene Ontology (GO) analysis of the AT2^Def cluster (Fisher’s Exact Test). j UMAP with overlaid DATP and Defender gene scores. k SPRING plot of clusters in (h), showing AT2^Def state (red) in between the control AT2 (orange) and AT2^DLK1+ (green) clusters. l SPRING plot of the DATP-gene score. m Immunostained PN3 mAT2 spheroids cultured with FGF7 alone (Control, top) or with interferon-γ (20 ng/mL; bottom). Bars, 20 µm. n Quantification of (m) showing percent of AT1s per spheroid (n represents spheroids sampled per condition). Data as mean ± SD (Student’s two-sided t test). o Schematic working model of AT2s oscillating between AT2^Def (purple) and AT2^DATP (yellow) states, both suppressed by C/EBPα. Upon AT2 loss (gray AT2s) and C/EBPα downregulation following infection, remaining AT2s could oscillate (left) between either state allowing for both a proper response to infection and regeneration. In a static model (right), inability to access both AT2^Def and AT2^DATP states would result in either impaired pathogen response or repair, respectively. All experiments were repeated at least three times (adapted from BioRender). Source data are provided as a Source Data file.