However, it is unclear whether the expression of AEC2 cell-specific proteins and transcription factors will change after PM2.5 stimulation. Canonical Wnt/-catenin signaling pathway is involved in stem cell proliferation and self-renewal regulation in many tissues40. function. Type 2 alveolar epithelial cells Imirestat (AEC2) are stem cells in the adult lung that contribute to the lung repair process through complex signaling. Our previous studies demonstrated that RAB6, a RAS family member lowly expressed in lung cancer, inhibited lung cancer stem cell self-renewal, but it is unclear whether or not and how RAB6 may regulate AEC2 cell proliferation and self-renewal in PM2.5-induced pulmonary fibrosis. Here, we demonstrated that knockout of RAB6 inhibited pulmonary fibrosis, oxidative stress, and AEC2 cell death in PM2.5-injured mice. In addition, knockout of RAB6 decreased Dickkopf 1(DKK1) autocrine and activated proliferation, self-renewal, and wnt/-catenin signaling of PM2.5-injured AEC2 cells. RAB6 overexpression increased DKK1 autocrine and inhibited proliferation, self-renewal and wnt/-catenin signaling in AEC2 cells in vitro. Furthermore, DKK1 inhibitors promoted proliferation, self-renewal and wnt/-catenin signaling of RAB6 overexpressing AEC2 cells, and attenuated PM2.5-induced pulmonary fibrosis in mice. These data Imirestat establish RAB6 as a regulator of DKK1 autocrine and wnt/-catenin signal that serves to regulate AEC2 cell proliferation and self-renewal, and suggest a mechanism that RAB6 disruption may promote AEC2 cell proliferation and self-renewal to enhance lung repair following PM2.5 injury. and diffusion capacity for carbon monoxide, forced expiratory volume in 1?s, forced vital capacity. Isolation, culture, and transfection of mouse AEC2 cells Mouse AEC2 cells were enriched by surface marker sorting as previously reported14. Fresh mouse lung tissues were digested with Dispase and Collagenase at 37?C for 20?min. Cells were resuspended and incubated with the antibody mixture anti-EPCAM(25C5791C80, eBioscience), anti-CD24(12C0242C82, eBioscience), anti-SFTPC(sc-518029, Santa Cruz), anti-CD31-CD34-CD45(13C0311C82, 13C0341C82, and 13-0451-82, eBioscience). The AEC2 cell population (CD24? SFTPC+ subset) was isolated from the epithelial cell populations (EPCAM+CD31?CD34?CD35?) by the FACSAria sorter. The sorted AEC2 cells were seeded in a matrigel 6-well plate (354671, Corning, USA) and cultured in bronchial epithelial cell growth medium (BEGM) supplied with 1% FBS Imirestat and growth factors (50?ng/mL FGF, 30?ng/mL HGF). The cell growth medium was changed every 2 days. For PM2.5 injury, WT and RAB6?/? AEC2 cells were exposed to PM2.5 (100?g/ml) or saline for 48?h as we previously described7. For cell transfection, the RAB6 overexpression vector was constructed and transfected into AEC2 cells by Lipofectamine 2000 as we previously described29. For DDK1 protein treatment, WT and RAB6?/? AEC2 cells were exposed to DKK1 protein (10?ng/ml) (ab205987, Abcam) or PBS for 48?h. For DKK1 inhibitor treatment, RAB6 overexpression (RAB6) and negative control (NC) AEC2 cells were exposed to DKK1 inhibitor (Gallocyanine, 5?M) or PBS for 48?h. Immunofluorescence Paraformaldehyde-fixed lung tissue or AEC2 cell samples were blocked and then incubated with primary antibodies RAB6 (9625, CST), SFTPC (sc-518029, Santa Cruz) or DKK1 (sc-374574, Santa Cruz) overnight. Next, the samples were incubated with FITCClabeled goat anti-rabbit antibody (31635, Invitrogen) and Alexa 647-conjugated goat anti-mouse antibody (A-21235, Imirestat Invitrogen). Nuclear staining was performed with DAPI stain solution. Confocal images were captured using a Leica TCS SP8 confocal microscope. RNA isolation and quantitative real-time PCR (qRT-PCR) Lung tissue or cells were lysed by TRIzol kit (QIAGEN) and RNA was isolated per the manufacturers instructions. In addition, the PCR was carried out by the One Step TB Green RT-PCR Kit (TaKaRa, Japan) as previously described. The relative expression of each gene was calculated using the 2 2?CT method after correction by GAPDH expression. All primer sequences are listed in TIMP2 the Supplemental Table 1. Histopathological analysis and immunohistochemistry Lung tissues of all mice fixed in paraformaldehyde and embedded in paraffin were sectioned to a thickness of 5?m. Then, the tissue slides were deparaffinized and rehydrated. For lung collagen detection, the tissue slides were stained with MASSON trichrome stain kit as previously described9. After MASSON staining, the slides were dehydrated in gradient alcohol, sealed, and photographed under a light microscope. For immunohistochemical detection of -SMA or RAB6, the tissue slides were subjected to antigen retrieval, and the endogenous peroxidase was inactivated by treatment with H2O2 (3%). The slides were incubated with -SMA (19245, CST) or RAB6 (9625, CST) antibodies, then washed and incubated with Biotin-labeled goat anti- rabbit IgG (65C6140, Invitrogen), followed by staining with the DAB.