Fig. 7

Nintedanib regulates the pool size of fibrocytes in bleomycin (BLM)-treated mouse lungs. Normal saline (NS) or BLM-treated mice with or without nintedanib (60 mg/kg/day) were killed on day 7. Paraffin-embedded lung sections were stained with a rabbit anti-S100A4/FSP-1 antibody (green) and anti-CD45 (red), and lung digests stained for collagen-1, CD45, and CXCR4 were examined by flow cytometry. Lung digests were also stained for isotype control IgG labeled with FITC, phycorythrin, or phycoerythrin-cyanine. a Representative images of immunohistochemical staining in each group are shown. Arrows indicate fibrocytes doubled stained for S100A4/FSP-1 and CD45 (scale bar = 100 μm). b After lung digests were first gated by collagen-1 as shown in above figures, only collagen-1+ cells were examined for dual expression of CD45 and CXCR4 using the logical gates depicted as shown in below figures. Collagen-1⁺ cells in lung digests were examined for the dual expression of CD45 and CXCR4 using the logical gates depicted. Collagen-1⁺CD45⁺CXCR4⁺ fibrocytes in lung digests from mice in each group were examined. c S100A4/FSP-1 and CD45 double-positive cells were counted in 10 random fields per section at 20× magnification for three separate lung section. Data were analyzed using a one-way ANOVA and displayed as dot plot and their means (n = 30 in each group). d The percentage of fibrocytes relative to all cells in lung digests were examined. Data were analyzed using the Kruskal-Wallis H test and displayed as median and interquartile range of four separate experiments. In all graphs: *P < 0.01 versus the group treated with BLM alone