Bone morphogenetic protein (BMP)4 promotes mitophagy and restores mitochondrial dynamics in transforming growth factor (TGF)-β1-stimulated lung fibroblasts. a) Representative Western blot results of Beclin-1, p62, LC3B and Pink1 protein expression in total cell lysates of primary lung fibroblasts from BMP4+/+ and BMP4+/− mice treated with TGF-β1 (10 ng·mL−1, 48 h) (n=4). β-actin was used as a loading control. b) Representative Western blot results of mitofusin (MFN)1, MFN2, dynamin-related protein (DRP)1 and mitochondrial fission 1 protein (FIS1) expressions in total cell lysates of primary lung fibroblasts from BMP4+/+ and BMP4+/− mice treated with TGF-β1 (10 ng·mL−1, 48 h) (n=4). β-actin was used as a loading control. c) Representative Western blot results of MFN1, MFN2, DRP1 and FIS1 expressions in total cell lysates of wild-type (WT) mouse primary lung fibroblasts treated with TGF-β1 (10 ng·mL−1, 48 h) and/or BMP4 (20 μM) (n=4). β-actin was used as a loading control. d) Mitochondrial fission was visualised by MitoTracker Red staining. Scale bars=25 µm. e) Immunofluorescence analysis of LC3B (red) and mitochondria (MitoTracker, green) in primary lung fibroblasts (n=4). Scale bars=25 µm. After Pink1 short-hairpin (sh)RNA (shPink1) or control shRNA (shNC) was transfected into NIH/3T3 fibroblasts, Pink1 protein expression was measured using f) Western blotting and g) immunofluorescence assays (n=4). Scale bars=100 µm. After shPink1 or shNC was transfected into NIH/3T3 fibroblasts for 24 h, cells were treated with TGF-β1 (10 ng·mL−1) and/or BMP4 (20 μM) for 48 h. h) Col1 and α-smooth muscle actin (SMA) protein expressions were examined with Western blot (n=4). i–k) The expression of α-SMA, fibronectin (FN) and Col1 was visualised using confocal microscopy (n=4). Scale bars=100 µm. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.