Fig. 5

Blockade of Wnt proteins attenuates myofibroblastic differentiation of LR-MSCs and pulmonary fibrosis. a–c Mice (n = 6 per group) were intraperitoneally injected with vehicle (10% DMSO/saline) or 5 mg/kg salinomycin every other day as indicated 7 days after bleomycin (BLM) instillation. Mice were killed on day 14 after BLM administration. a Cytoplasmic and nuclear extracts were prepared and analyzed by western blot using anti-p-β-catenin and anti-β-catenin antibodies. Lamin B was used as the nuclear internal control, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the cytoplasmic internal control. b The expression of α-smooth muscle actin (α-SMA) and collagen I in mouse lung tissues was analyzed by immunofluorescence. c The protein levels of stem cell antigen-1 (Sca-1) in mouse lung tissues were determined by western blot. d Mouse lung-resident mesenchymal stem cells (LR-MSCs) were pretreated with 1 μM salinomycin for 1 h followed by stimulation with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 48 h. The expression of α-SMA, collagen I and fibronectin was examined by western blot. e–g Mice (n = 6 per group) received either LV-negative control (NC) or LV-Wnt10a-siRNA (2 × 10⁸ TU in 50 μl of saline) intratracheally on day 3 after BLM instillation. Mice were killed 14 days after BLM administration. e The effect of LV-Wnt10a-siRNA on pulmonary fibrotic lesions was determined by hematoxylin-eosin (H&E) staining. f The expression of the fibrosis markers and Sca-1 in mouse lung tissues was measured by western blot. g Cytoplasmic and nuclear extracts were prepared and analyzed by western blot using anti-p-β-catenin and anti-β-catenin antibodies. Lamin B and GAPDH were used as the nuclear and cytosolic markers, respectively. h–j Mouse LR-MSCs were transfected with LV-NC or LV-Wnt10a-siRNA in the presence or absence of 10 ng/ml TGF-β1 followed by incubation for 48 h. h The expression of p-β-catenin and β-catenin in the cytoplasm and nucleus was examined by western blot. Lamin B and GAPDH were used as the loading controls for nuclear and cytoplasmic proteins, respectively. i The protein levels of the fibrosis markers in mouse LR-MSCs were measured by western blot. j The expression of α-SMA in mouse LR-MSCs was analyzed by immunofluorescence