Fig. 5

The essential role of β-catenin in maintaining the expression of alveolar type 2 (AT2) markers. A A retroviral vector expressing three siRNAs against mouse β-catenin was stably introduced into imPAC2 cells to generate the imPAC2-simBC cells. The qPCR analysis confirmed that β-catenin was effectively silenced in the imPAC2-simBC cells, compared with that in the imPAC2 cells (** p < 0.01). B Silencing β-catenin leads to the decreased expression of AT2 markers in vitro. Total RNA was isolated from subconfluent imPAC2 and imPAC2-simBC cells and subjected to qPCR analysis of the expression of Nkx2.1, SftpA, SftpB, SftpC, SftpD, and Abca3. “**” p < 0.01, compared with that of the imPAC2 group. C & D Silencing β-catenin leads to the decreased expression of AT2 markers in vivo. Subconfluent imPAC2 and imPAC2-simBC cells were collected, resuspended in scaffold material PPCNg/PBS, and subcutaneously injected into the flanks of athymic nude mice (5 × 10⁶ cells per injection). At 30 days after implantation, the subcutaneous masses were retrieved from the injection sites, fixed with paraformaldehyde, and subjected to H & E staining, and immunofluorescence staining with anti-SftpC antibody (C). Cell nuclei were counterstained with DAPI. Staining without primary antibodies was used as a negative control (not shown). Representative images are shown (C). Total RNA was also isolated from the freshly retrieved masses and subjected to qPCR analysis of the expression of Nkx2.1, SftpB, SftpC, and SftpD (D). Gapdh was used as a reference gene. “**” p < 0.01, compared with that of the imPAC2 cell group