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. 2024 Oct 24;15:9169. doi: 10.1038/s41467-024-53330-1

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A Experimental scheme for the bleomycin-induced lung injury model. Pclaf WT and KO mice were treated with PBS (n = 5 for Pclaf WT, n = 5 for Pclaf KO) or bleomycin (1.4 U/kg; n = 25 for Pclaf WT, n = 24 for Pclaf KO; Bleo) by intratracheal instillation. The blood oxygen level (SpO₂) and breath rate were measured by pulse-oximetry at the indicated time points. At 3 days after injury (dpi, n = 7 for each group with bleomycin), 7 dpi (n = 8 for each group with bleomycin), and 21 dpi (n = 10 for Pclaf WT, n = 8 for Pclaf KO with bleomycin, n = 5 for each group with PBS (21 dpi)), the lungs were collected for further analysis. B The dynamics of SpO₂ levels (left panel) and breath rate per minute (right panel) were measured at the indicated time points. Pclaf WT and KO with PBS (n = 5); Pclaf WT with bleomycin until 3 dpi (n = 25); 5 dpi and 7 dpi (n = 18), after 9 dpi (n = 10); Pclaf KO with bleomycin until 3 dpi (n = 24); 5 dpi and 7 dpi (n = 17), 9–15 dpi (n = 9), after 17 dpi (n = 8). C Representative immunostaining images for RAGE (AT1) and CDH1 (Epithelial cell) in PBS-instilled lung and 7 dpi of bleomycin-instilled lung. D Representative images of immunostaining for RAGE and SPC (AT2 cells) at 7 dpi. E Quantification of RAGE⁺ area/CDH1⁺ area. Pclaf WT with PBS (n = 8), 3 dpi (n = 8), 7 dpi (n = 8), and 21 dpi (n = 10); Pclaf KO with PBS (n = 10), 3 dpi (n = 8), 7 dpi (n = 9), and 21 dpi (n = 10). F Quantification of SPC⁺ cells. n = 10 for each strain at indicated time point. G Representative images of hematoxylin and eosin (H&E) staining at 21 dpi. H Representative images of picrosirius staining (collagen fiber) at 21 dpi. I Representative images of immune-staining for alpha-smooth muscle actin (αSMA/Acta2; smooth muscle cell) at 21 dpi. J Quantification graphs of the picrosirius⁺ area. Pclaf WT or Pclaf KO with PBS (n = 5) or bleomycin at 21 dpi (n = 10). K Quantification graphs of the αSMA⁺ area. n = 12 for each strain. L Quantification of hydroxyproline contents in the left lobe of bleomycin (n = 5) or PBS-instilled (n = 3) lung at 25 dpi. M, N The lung epithelial cells were isolated from bleomycin-treated lungs of Pclaf WT or Pclaf KO mice at 7 dpi by MACS. The lung epithelial cells (TER119^-/CD31^-/CD45^-/EPCAM⁺) were cultured with lung endothelial cells (CD31⁺) at a liquid-air interface to generate LOs. Representative Images of alveolar type organoids fluorescently immunostained for HOPX (AT1) and SPC (M). Quantification graph of HOPX⁺ and SPC⁺ cells. Pclaf WT (n = 9) or Pclaf KO (n = 8) (N). Two-sided Student’s t-test; error bars: mean +/− SD. Represented images are shown (n ≥ 3). Source data are provided as a Source data file.