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. 2023 May 4;14:2560. doi: 10.1038/s41467-023-38177-2

Fig. 8. Endothelial-specific overexpression of FOXF1 during fibrotic stage decreases lung fibrosis and increases survival of mice after bleomycin-induced injury.

Fig. 8

a Schematic diagram shows bleomycin, tamoxifen (TAM) and doxycycline (DOX) treatment in endFoxf1OE mice. b Endothelial-specific overexpression of FOXF1 decreases bodyweight loss in bleomycin-treated endFoxf1OE mice (Control, n = 12; endFoxf1OE, n = 12 for days 0 and 21, n = 4 for day 10, n = 6 for day 14), *p < 0.05, two-way ANOVA with the Geisser–Greenhouse correction. c Endothelial-specific overexpression of FOXF1 increases mouse survival after bleomycin injury (Control, n = 11; endFoxf1OE, n = 17), *p < 0.05, Log-rank (Mantel–Cox) test. d Decreased collagen depositions in bleomycin-treated endFoxf1OE lungs is shown by Hydroxyproline assay. Left lung lobes from endFoxf1OE (n = 4) and control (n = 3) mice were used, **p = 0.0061, t test (two tailed). e Decreased fibrosis in bleomycin-treated endFoxf1OE lungs is shown by H&E, Sirius red/fast green, Trichrome, and immunostaining for αSMA. Lungs were harvested at day 21 after bleomycin treatment (n = 8 per group). Bar = 100 µm. fi Ashcroft score of endFoxf1OE lungs (***p < 0.0001) is consistent with the percent of Sirius red-positive (***p < 0.0001), Trichrome-positive (**p = 0.0031) and αSMA-positive areas (***p < 0.0001). Areas positive for collagen depositions were quantified in 10 random fields per lung using Nikon NIE CIC Analysis Elements software. n = 8 mice per group. j Endothelial overexpression of Foxf1 at day 10 after bleomycin administration decreases mRNA levels of pro-fibrotic genes in total lung RNA as shown by qRT-PCR. Total lung mRNA was extracted from control and bleomycin-treated endFoxf1OE mice on day 21. Actb mRNA was used for normalization (Control Col1a1 and Col3a1, n = 10; Control Vim, n = 9; Control Cthrc1, n = 9; Control Fn1, n = 8; endFoxf1OE Col1a1, Col3a1, Vim and Cthrc1, n = 7; endFoxf1OE Fn1, n = 8). k Decreased number of macrophages in the lungs of bleomycin-treated endFoxf1OE mice is shown using immunofluorescent staining with anti-F4/80 antibodies (red) and anti-αSMA antibodies (green) at day 21 after bleomycin administration, ***p < 0.0001. Nuclei are counterstained with DAPI (blue). n = 8 mice per group. Data presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test (two tailed). Source data are provided as a Source Data file.