Research Paper Volume 12, Issue 1 pp 242—259

Epithelial cell senescence induces pulmonary fibrosis through Nanog-mediated fibroblast activation

Figure 3. Epithelial cell senescence could induce pulmonary fibroblast activation via activating Wnt/β-catenin signalling. (AC) MLE-12 cells were treated with bleomycin (BLM, 25 μg/ml) for the indicated times. (A, B) SA-β-gal staining and β-galactosidase activity measurement were performed to detect cellular senescence, * P < 0.05 and ** P < 0.01 vs. 0 h. (C) The protein levels of p21 and p16 were measured by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01. (DI) MLE-12 cells were pre-treated with or without BLM for 3 days. The medium was replaced by fresh medium without BLM and co-cultured with pulmonary fibroblasts for another 3 days. (D) The migration capacity of pulmonary fibroblasts was detected by using a wound-healing assay. Wound areas were calculated by ImageJ, **P < 0.01. (E) The proliferation ability of pulmonary fibroblasts was measured by EdU assay. The percentage of proliferating cells were calculated by ImageJ, **P < 0.01. (F) The protein levels of collagen I, vimentin and α-SMA were determined by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, **P < 0.01. (G) Pulmonary fibroblasts were double stained with vimentin and collagen I by immunofluorescence. (H) Pulmonary fibroblasts were double stained with α-SMA and periostin by immunofluorescence. (I) The expression of β-catenin was measured by immunofluorescence. (J) MLE-12 cells were pre-treated with or without BLM for 3 days. MLE-12 cells were cultured with fresh medium without BLM for another 3 days. The supernatants were collected to culture pulmonary fibroblasts in the presence or absence of ICG-001. The expression of β-catenin, α-SMA and collagen I were examined by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01.