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. 2023 Feb 13;4(3):100945. doi: 10.1016/j.xcrm.2023.100945

Figure 1.

Figure 1

Low-dose bleomycin induces irreversible pulmonary fibrosis in aged mice

(A) Line plot showing body weights of aged wild-type (WT; C57BL/6J) mice 7, 14, and 21 days after intratracheal administration of normal saline (equal volume) or bleomycin (1 U/kg body weight) (n = 8 WT saline; n = 8 WT bleomycin). ∗∗∗p < 0.001, two-way ANOVA with Tukey’s post-hoc test.

(B) Representative images of Mason’s trichrome-stained lung sections of aged mice 28 or 50 days after intratracheal administration of bleomycin. Scale bars, 50 μm (top) and 1 mm (bottom) (n = 8 WT saline; n = 8 WT bleomycin).

(C) Hydroxyproline content (μg per mg of lung tissue) in the lungs of aged mice 28 or 50 days after intratracheal administration of bleomycin (1 U/kg body weight). ∗∗∗p < 0.001, ∗p < 0.05, one-way ANOVA with Tukey’s post-hoc test.

(D) Western blot for expression of IGFBP2, P21, collagen-1, fibronectin, and vimentin in the lung homogenates of aged mice 14 days after low-dose bleomycin challenge. β-actin served as the internal control. Note that same samples were run on different gels (n = 6 WT saline; n = 8 WT bleomycin).

(E) Igfbp2 mRNA expression in the lung homogenates of aged mice subjected to low-dose bleomycin challenge after 14 days.

(F) IGFBP2 protein expression was determined by ELISA in the serum samples of aged mice 14 days after bleomycin injury (n = 5 WT saline; n = 6 WT bleomycin).

(G) Igfbp2 mRNA expression in the primary AEC2 cells isolated from aged mice subjected to low-dose bleomycin challenge after 14 days. Each sample is obtained from 4 mice lungs (n = 3 WT saline; n = 3 WT bleomycin). NS, not significant, ∗∗∗∗p < 0.0001, ∗∗p < 0.01, Student’s unpaired two-tailed t test.

(H) Representative multicolor immunohistochemistry of lung sections from aged WT mice subjected to low-dose bleomycin after 28 and 50 days. Green color indicates surfactant protein C (SPC) expression; brown color indicates P21 (top) and phospho-H2AX (bottom) expression. Insets: zoom-in images to show green and brown color localization. Black arrowheads highlight the double-positive AEC2 cells. Scale bars, 10 μm.

(I and J) Quantification of double-positive cells for P21 (left) and phospho-H2AX (right) expression in SPC + cells in the lungs of aged WT mice subjected to low-dose bleomycin after 28 and 50 days, respectively. Each data point represents per field image of a sample. Data are mean ± SEM. ∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05, one-way ANOVA with Tukey’s post-hoc test.