Fig. 3

Selection and characteristics of the EpCAM⁺ cells of immortalized mouse alveolar type 2 cells (imPAC2). A FACS analysis of the isolated imPAC2 cells. The pooled imPACs were subjected to FACS sorting analysis with an EpCAM antibody; and the EpCAM⁺ cells were enriched with streptavidin-coated microbeads (Miltenyi Biotec), resulting in the imPAC2 cells (Additional file 1: Figure S1D), which were further confirmed for the presence of the AT2 marker pro-SPC with > 97% positivity. B Western blotting analysis of SftpA, SftpB, SftpC and SftpD expression in imPAC2 cells. The imPAC2 cells (a) and imPAC2 culture supernatant (b) were subjected to Western blotting using SftpA, SftpB, SftpC and SftpD antibodies, as well as long with β-actin antibody for internal loading control. C The qPCR-based expression analysis of AT2 markers SftpA, SftpB SftpC and SftpD expression in imPAC2 cells. Total RNA was isolated from subconfluent primary mPAC cells, imPACs and imPAC2 cells; and subjected to RT-qPCR analysis using gene-specific qPCR primers for AT2 makers SftpA, SftpB, SftpC and SftpD. Gapdh was used as a reference gene to normalize the cDNA levels among the samples. “**” p < 0.01, “*” p < 0.05, compared with that of the mPACs group. D Immunofluorescence analysis of SftpA, SftpB, SftpC and SftpD expression in imPAC2 cells. Subconfluent imPAC2 cells were subjected to immunostaining with primary antibodies against SftpA, SftpB, SftpC, SftpD. Cell nuclei were counterstained with DAPI. Representative images are shown. Staining without primary antibodies was used as a negative control (data not shown)