Fig. 2

Evaluation of ITGA8 as a cell surface marker for lung lipofibroblasts. (A) FACS panels of PDGFRα⁺ lung fibroblasts and LipidTOX-fluorescence intensities between SCA-1-single-positive, ITGA8-single-positive, and double-positive fibroblasts. The right graph is the quantification. Data represent the mean ± SEM of results obtained from three independent adult male mice (wild-type). *P < 0.05 (one-way analysis of variance with Bonferroni correction). Lineage = a mixture of EpCAM, CD31, CD45, CD146, and LYVE-1. Note: ITGA8 was also expressed in non-fibroblast cells (CD31-positive cells: 5.2% in average; CD45-positive cells: 1.9%; CD146-positive cells: 4.4%; EpCAM-positive cells: 0.7%) (n = 3, wild-type adult male mice). (B) Representative images of Oil Red O staining with hematoxyline for SCA-1-single-positive and ITGA8-single-positive lung fibroblasts (scale bar: 50 μm) and the quantification including double-positive fibroblasts (sum and breakdown of the scores). Data represent the mean ± SEM of results obtained from three independent adult male mice (wild-type). **P < 0.01, ***P < 0.001 (one-way analysis of variance with Bonferroni correction). (C) Representative images of LipidTOX staining with DAPI (scale bar: 100 μm) for SCA-1-single-positive and ITGA8-single-positive lung fibroblasts and the quantification including double-positive fibroblasts (sum and breakdown of the scores). Data represent the mean ± SEM of results obtained from three independent adult male mice (wild-type). ***P < 0.001 (one-way analysis of variance with Bonferroni correction). (D) Representative images of newborn (P7), adult (2 months), and aged (2 years) lungs stained with LipidTOX, anti-ITGA8 antibody, and DAPI (scale bars: 20 μm), and the quantification of LipidTOX-fluorescence intensities of ITGA8⁺ lung fibroblasts evaluated by flowcytometry. Triangles show ITGA8⁺LipidTOX⁺ fibroblasts. Data represent the mean ± SEM of results obtained from three independent mice (wild-type) in each generation. **P < 0.01 (one-way analysis of variance with Bonferroni correction)