Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 2. Generation of FASN transgenic mice. (A) Constructs for generation of FASN transgenic mice. (B) Immunoblotting analysis of human FASN- His in control and doxycycline-treated mice. FASN antibody detects both humans and mice FASN protein. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 5. (C) Immunohistochemical stain of the lungs of wild type, control and doxycycline-treated FASN transgenic mice. Scale bar, 50 µm.

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Transgenic Assay, Construct, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 1. FASN protein levels were decreased in the lungs of patients with IPF and in BLM-treated mice. (A) Immunohistochemical staining of FASN in human and mouse lungs. FASN was strongly localized in AECs (inset) in NC or control mice. Original magnification: × 100, inset panels × 400. (B) Lung FASN expression was measured by immunoblotting. Densitometry of FASN bands. Equal amounts of protein obtained from lung lysates were subjected to immunoblotting with anti–FASN Ab. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 5. The data are expressed as the mean ± standard error of the mean. The data were normalized to β-actin. *P < 0.05, vs. NC or CTL. (C) Historical analysis of combination of immunofluorescence staining with anti-FASN and anti–SPC in IPF and NC (upper panels, n = 6 per group), CTL and BLM (lower panels, n = 6 per group). SPC-positive cells (green), FASN cells (red), and DAPI (blue) are shown. (D) Representative immunofluorescence images showing FASN (red) in AECIIs (using anti‐SPC, green) of NC, IPF lungs (upper panels, n = 6 per group), CTL and BLM lungs (lower panels, n = 6 per group). Scale bar, 50 µm. The data are expressed as the mean ± standard error of the mean. *P < 0.05, vs. NC or CTL.

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 3. Effects of knockdown or overexpression of FASN on death of A549 alveolar epithelial cells and in the lungs of FASN transgenic mice. Stable FASN-overexpressing or FASN-knockdown cells were treated with BLM (10 μg/mL) for 24 h in serum-free medium. (A) Apoptosis was defined by annexin V+/both PI+ and PI− staining. Necrosis was defined by PI+/both Annexin V+ and Annexin V− staining. Quantification of the apoptotic and necrotic cell percentage. The data are expressed as the mean ± SEM (n = 4). *P < 0.05, vs. scramble or pCMV. # P < 0.05, vs. scramble + BLM or pCMV + BLM. (B) Protein expression of the total and active forms of caspase-3 was measured by immunoblotting. The intensity of the active form of caspase-3 was quantified by densitometry, and the data were normalized to β-actin. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 5. The data are expressed as the mean ± SEM. Error bars represent mean ± SEM (n = 4) *P < 0.05, vs. scramble or pCMV. # P < 0.05, vs. scramble + BLM or pCMV + BLM. (C) Tissues stained by the TUNEL method were observed at × 100 magnification, and the number of TUNEL-positive cells in 20 fields per lung was counted (n = 8/group). The data are expressed as the mean ± SEM. * P < 0.05, vs doxy(−)/BLM (+).

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Knockdown, Over Expression, Transgenic Assay, Staining, Expressing, Western Blot, TUNEL Assay

Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 4. FASN regulates BLM-induced loss of mitochondrial membrane potential and mitochondrial dysfunction in AECs. FASN-overexpressing and knockdown cells treated with BLM (10 μg/mL) were stained with JC-1 for 30 min and analyzed by flow cytometry. (A) The proportion of depolarizing mitochondrial membrane potential (JC-1 green fluorescence) was increased in FASN knockdown cells, whereas it was decreased by FASN overexpression. Graph showing the ratio of the intensity of red to green fluorescence (JC-1 aggregate and JC-1 monomer) of flow cytometry. The data are expressed as the mean ± standard error of the mean (n = 4). *P < 0.05, vs Scramble or pCMV. #P < 0.05, vs Scramble + BLM or pCMV + BLM. (B) Protein expression of PINK1 was measured by Immunoblotting analysis in the lungs of FASN transgenic mice. The intensity of the PINK1 was quantified by densitometry, and the data were normalized to β-actin. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 5. The data are expressed as the mean ± SEM. Error bars represent mean ± SEM (n = 6) *P < 0.05, vs. Doxy (−)/BLM (+). (C) Immunofluorescence analysis images of PINK1 (red) in AECIIs (SPC, green) of FASN transgenic mice along with the quantification of double positive cells (n = 6, each group). Scale bar, 50 µm. The data are expressed as the mean ± standard error of the mean. *P < 0.05, vs. Doxy (−)/BLM (−)panels × 400.

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Membrane, Knockdown, Staining, Flow Cytometry, Fluorescence, Over Expression, Expressing, Western Blot, Transgenic Assay, Immunofluorescence

Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 5. FASN regulates BLM-induced production of reactive oxygen species (ROS) in AECs. FASN overexpressing and knockdown cells were treated with BLM (10 μg/mL) for 24 h and analyzed by flow cytometry. Mitochondrial ROS production was detected with MitoSox-Red (red). The data are expressed as the mean ± standard error of the mean (n = 4). Scale bar, 50 µm. *P < 0.05, vs Scramble or pCMV. #P < 0.05, vs Scramble + BLM or pCMV + BLM.

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Knockdown, Flow Cytometry

Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 6. Fatty acid profiles of FASN overexpressing and knockdown cells and AECs from FASN transgenic mice, and beneficial effect of oleic acid on BLM -treated AECs. (A) Changes in fatty acid composition by overexpression or knockdown of the cells were determined by LC–MS. (B) Changes in fatty acid composition in AECs from FASN transgenic mice lung were determined by LC–MS. * < 0.05, Doxy(−)/BLM (−) vs Doxy (+)/BLM (−). #P < 0.05, Doxy (−)/BLM (+) vs Doxy (+)/BLM (+). (C) The WST-1 assay showed that treatment with oleic acid (100 μM) improved survival of BLM (10 μg/mL)-treated primary mouse AECs. (D) Treatment with oleic acid (100 nM) rescued AECs from BLM (10 μg/mL)-induced apoptotic cell death. Apoptosis was defined by Annexin V+/both PI+ and PI− staining. Necrosis was defined by PI+/both Annexin V+ and Annexin V− staining. The data are expressed as the mean ± standard error of the mean (n = 4). * < 0.05, BLM vs BLM + OA.

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Knockdown, Transgenic Assay, Over Expression, Liquid Chromatography with Mass Spectroscopy, WST-1 Assay, Staining

Journal: Scientific reports

Article Title: Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice.

doi: 10.1038/s41598-023-36009-3

Figure Lengend Snippet: Figure 7. FASN overexpression attenuates BLM- induced lung inflammation and fibrosis in a mouse model. (A) Protein expression of FASN was measured by immunoblotting. The intensity of FASN was quantified by densitometry, and the data were normalized to β-actin. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 5. The data are expressed as the mean ± SEM. Error bars represent mean ± SEM (n = 6) *P < 0.05, vs. Doxy (−)/BLM (+). (B) H&E staining of control and BLM-treated FASN transgenic mouse lungs. Original magnification × 10; original magnification × 100 (C) Cell counts in BALF, collected on day 21. The total number of cells was counted using a hemocytometer. Differential cell counts in BALF were analyzed from 500 cells stained with Diff-Quick (n = 8 per group). (D) Masson’s trichrome staining in control and BLM-treated FASN transgenic mouse lungs. Original magnification × 200. (E) Quantification of lung fibrosis by Ashcroft score (n = 8 per group). Data are expressed as the mean ± SEM. (F) Collagen measurement by the hydroxyproline assay of control and BLM-treated mouse lungs (n = 8 per group). (G) Active TGF-β1 levels in control and BLM-treated mouse lungs (n = 8 per group). TGF-β1 levels in lung lysates were measured by ELISA. *P < 0.05 CTL/BLM(+) vs. FASN TG/BLM(+).

Article Snippet: We also performed lentiviral-mediated infection of A549 cells with human FASN shRNA (no. TL31058V; OriGene, Rockville, MD, USA), which was shown to efficiently knock down human FASN protein expression (supplementary Fig. 1B).

Techniques: Over Expression, Expressing, Western Blot, Staining, Control, Transgenic Assay, Diff-Quik, Hydroxyproline Assay, Enzyme-linked Immunosorbent Assay