Journal: Cells

Article Title: Trehalose Alleviates Crystalline Silica-Induced Pulmonary Fibrosis via Activation of the TFEB-Mediated Autophagy-Lysosomal System in Alveolar Macrophages

doi: 10.3390/cells9010122

Figure Lengend Snippet: TFEB overexpression relieves CS-induced macrophage apoptosis and inflammation via autophagic substrate degradation in vitro. ( A , B ) Immunoblotting analysis of LC3II, Atg5, and Beclin 1 protein levels ( n = 4). ( C , D ) Immunoblotting analysis of LAMP1 protein levels ( n = 3). ( E ) Cells were grown on coverslips and treated with CS followed by staining with 50 nmol/L Lysotracker Red or 5 μg/mL acridine orange at 37 °C for 30 min (scale bar = 10 μm and n = 4). ( F ) Immunofluorescence analysis of the colocalization of LAMP1 and LC3 12 h post CS treatment (scale bar = 25 μm and n = 4). ( G , H ) Immunoblotting analysis of p62 protein levels ( n = 4). ( I ) Immunofluorescence analysis of the colocalization of the p62 and ubiquitin 12 h post CS treatment (scale bar = 25 μm and n = 4). (J , K ) TUNEL (green) and DAPI (blue) staining and ratios of TUNEL-positive apoptotic cells (scale bar = 50 μm and n = 4). ( L – O ) ELISA analysis of IL-6, MCP-1, TNF-α, and IL-1β levels in MH-S cell supernatant ( n = 3 to 4). Data are presented as the mean ± SD. *, p < 0.05 and **, p < 0.01.

Article Snippet: The following antibodies were used for immunofluorescence (IF), immunoblotting (IB), or immunohistochemistry (IHC) in preset study: β-Actin (4967, Cell Signaling Technology [CST], Danvers, MA, USA, 1:1000 for IB); lamin B (12987-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); F4/80 (sc-377009, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:50 for IF); TFEB (A303-673A-M, Bethyl Laboratories, Montgomery, AL, USA, 1:1000 for IB and 133721-1-AP, Proteintech, Philadelphia, PA, USA, 1:100 for IF, 1:200 for IHC); LC3 (12741, CST, Danvers, MA, USA, 1:1000 for IB and PM036, Medical & Biological Laboratories [MBL], Aichi, Japan, 1:200 for IF); Atg5 (129994, CST, Danvers, MA, USA, 1:1000 for IB); ubiquitin (10201-2-AP, Proteintech, Philadelphia, PA, USA, 1:500 for IB and sc-8017, Santa, Santa Cruz, CA, USA;1:50 for IF); caspase-3 (Cas3) (19677-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); BAX (50599-1-lg, Proteintech, Philadelphia, PA, USA, 1:500 for IB), BCL-2 (12789-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); cathepsin B (CTSB) (31718, CST, Danvers, MA, USA, 1:1000 for IB); LAMP1 (ab24170, abcam, Cambridge, UK, 1:1000 for IB and 1:200 for IF); collagen I (Col-1) (WL0088, Wanleibio, Liaoning, China, 1:500 for IB and abs131984, Absin Bioscience, Shanghai, China, 1:100 for IHC); fibronectin (Fn) (WL03180, Wanleibio, Liaoning, China, 1:500 for IB and NBP1-91258ss, Novus biological, Littleton, CO, USA, 1:200 for IHC); Beclin 1 (3738S, CST, Danvers, MA, USA, 1:1000 for IB); p62 (ab109012, abcam, Cambridge, UK, 1:10000 for IB); peroxidase-conjugated goat anti-rabbit IgG (H+L) (ZB2301, ZSGB-BIO, Beijing, China, 1:5000 for IB); rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H+L) (ZB0316, ZSGB-BIO, Beijing, China, 1:200 for IF); fluorescein-conjugated goat anti-rabbit IgG (H+L)(ZF0311, ZSGB-BIO, Beijing, China, 1:200 for IF); rhodamine (TRITC)-conjugated goat anti-mouse IgG (H+L) (ZF-0313, ZSGB-BIO, Beijing, China, 1:200 for IF); and Alexa Fluor ® 488 AffiniPure Fab Fragment Goat Anti-rabbit IgG (H+L) (111-547-003, Jackson ImmunoResearch, West Grove, PA, USA, 1:100 for IF).

Techniques: Over Expression, In Vitro, Western Blot, Staining, Immunofluorescence, Ubiquitin Proteomics, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Trehalose Alleviates Crystalline Silica-Induced Pulmonary Fibrosis via Activation of the TFEB-Mediated Autophagy-Lysosomal System in Alveolar Macrophages

doi: 10.3390/cells9010122

Figure Lengend Snippet: Trehalose (Tre) affects autophagy-associated proteins in the lungs and AMs of CS-treated mice. ( A , B ) Immunoblotting analysis of cytosol and nuclear TFEB levels in lung tissue on day 7 or 56 post CS or Tre treatment; β-actin, loading control for cytosolic proteins; and LaminB, loading control for nuclear proteins ( n = 3 to 4). ( C ) qPCR analysis of TFEB levels in lung tissue on day 7 or 56 post-CS or Tre treatment ( n = 4). ( D ) Immunofluorescence analysis of TFEB nuclear translocation in primary AMs in BALF on day 7 or 56 (scale bar = 10 μm and n = 3 to 4). ( E ) qPCR analysis of TFEB expression in AMs on day 7 or 56 post CS or Tre treatment ( n = 4). ( F , G ) Immunoblotting analysis of LC3II, Atg5, and Beclin 1 protein levels in lung tissue on day 7 or 56 post CS and Tre treatment ( n = 5 to 6). ( H ) Immunofluorescence analysis of LC3 in AMs on day 7 or 56 post CS or Tre treatment (Scale bar = 25 μm and n = 3). ( I , J ) Immunoblotting analysis of LC3II and Atg5 protein levels in AMs on day 7 or 56 post CS or Tre treatment ( n = 3 to 4). Data are presented as the mean ± SD. *, p < 0.05; **, p < 0.01; and ##, p < 0.01.

Article Snippet: The following antibodies were used for immunofluorescence (IF), immunoblotting (IB), or immunohistochemistry (IHC) in preset study: β-Actin (4967, Cell Signaling Technology [CST], Danvers, MA, USA, 1:1000 for IB); lamin B (12987-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); F4/80 (sc-377009, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:50 for IF); TFEB (A303-673A-M, Bethyl Laboratories, Montgomery, AL, USA, 1:1000 for IB and 133721-1-AP, Proteintech, Philadelphia, PA, USA, 1:100 for IF, 1:200 for IHC); LC3 (12741, CST, Danvers, MA, USA, 1:1000 for IB and PM036, Medical & Biological Laboratories [MBL], Aichi, Japan, 1:200 for IF); Atg5 (129994, CST, Danvers, MA, USA, 1:1000 for IB); ubiquitin (10201-2-AP, Proteintech, Philadelphia, PA, USA, 1:500 for IB and sc-8017, Santa, Santa Cruz, CA, USA;1:50 for IF); caspase-3 (Cas3) (19677-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); BAX (50599-1-lg, Proteintech, Philadelphia, PA, USA, 1:500 for IB), BCL-2 (12789-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); cathepsin B (CTSB) (31718, CST, Danvers, MA, USA, 1:1000 for IB); LAMP1 (ab24170, abcam, Cambridge, UK, 1:1000 for IB and 1:200 for IF); collagen I (Col-1) (WL0088, Wanleibio, Liaoning, China, 1:500 for IB and abs131984, Absin Bioscience, Shanghai, China, 1:100 for IHC); fibronectin (Fn) (WL03180, Wanleibio, Liaoning, China, 1:500 for IB and NBP1-91258ss, Novus biological, Littleton, CO, USA, 1:200 for IHC); Beclin 1 (3738S, CST, Danvers, MA, USA, 1:1000 for IB); p62 (ab109012, abcam, Cambridge, UK, 1:10000 for IB); peroxidase-conjugated goat anti-rabbit IgG (H+L) (ZB2301, ZSGB-BIO, Beijing, China, 1:5000 for IB); rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H+L) (ZB0316, ZSGB-BIO, Beijing, China, 1:200 for IF); fluorescein-conjugated goat anti-rabbit IgG (H+L)(ZF0311, ZSGB-BIO, Beijing, China, 1:200 for IF); rhodamine (TRITC)-conjugated goat anti-mouse IgG (H+L) (ZF-0313, ZSGB-BIO, Beijing, China, 1:200 for IF); and Alexa Fluor ® 488 AffiniPure Fab Fragment Goat Anti-rabbit IgG (H+L) (111-547-003, Jackson ImmunoResearch, West Grove, PA, USA, 1:100 for IF).

Techniques: Western Blot, Control, Immunofluorescence, Translocation Assay, Expressing

Journal: Cells

Article Title: Trehalose Alleviates Crystalline Silica-Induced Pulmonary Fibrosis via Activation of the TFEB-Mediated Autophagy-Lysosomal System in Alveolar Macrophages

doi: 10.3390/cells9010122

Figure Lengend Snippet: Tre relieves CS-induced lysosome damage and restores autophagic substrate degradation through TFEB activation in vitro. ( A , B ) Immunoblotting analysis of TFEB levels in the cytosol and nucleus of MH-S cells post CS and Tre treatment and β-actin, loading control for cytosolic proteins; LaminB, loading control for nuclear proteins ( n = 3 to 4). ( C ) qPCR analysis of TFEB expression in MH-S cells 12 h post-CS and Tre treatment ( n = 3 to 4). ( D ) MH-S cells were transfected with the adenovirus mRFP-GFP-LC3 plasmid. LC3 puncta were observed by confocal microscopy (scale bar = 10 μm and n = 3). ( E , F ) Immunoblotting analysis of LC3II, Atg5, and Beclin 1 protein levels ( n = 3). ( G , H ) Immunoblotting analysis of LAMP1 protein levels in cell lysates ( n = 4). ( I ) Cells were grown on coverslips and stained with Lysotracker Red or acridine orange 12 h post CS and Tre treatment (scale bar = 10 μm and n = 3). ( J ) Immunofluorescence analysis of the colocalization of LAMP1 and LC3 12 h post CS and Tre treatment (scale bar = 25 μ and n = 3). ( K – M ) Immunoblotting analysis of p62 and immunofluorescence analysis of the colocalization of p62 and ubiquitin 12 h post CS and Tre treatment (scale bar = 25 μm and n = 3). Data are presented as the mean ± SD. *, p < 0.05; **, p < 0.01; #, p < 0.05; and ##, p < 0.01.

Article Snippet: The following antibodies were used for immunofluorescence (IF), immunoblotting (IB), or immunohistochemistry (IHC) in preset study: β-Actin (4967, Cell Signaling Technology [CST], Danvers, MA, USA, 1:1000 for IB); lamin B (12987-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); F4/80 (sc-377009, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:50 for IF); TFEB (A303-673A-M, Bethyl Laboratories, Montgomery, AL, USA, 1:1000 for IB and 133721-1-AP, Proteintech, Philadelphia, PA, USA, 1:100 for IF, 1:200 for IHC); LC3 (12741, CST, Danvers, MA, USA, 1:1000 for IB and PM036, Medical & Biological Laboratories [MBL], Aichi, Japan, 1:200 for IF); Atg5 (129994, CST, Danvers, MA, USA, 1:1000 for IB); ubiquitin (10201-2-AP, Proteintech, Philadelphia, PA, USA, 1:500 for IB and sc-8017, Santa, Santa Cruz, CA, USA;1:50 for IF); caspase-3 (Cas3) (19677-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); BAX (50599-1-lg, Proteintech, Philadelphia, PA, USA, 1:500 for IB), BCL-2 (12789-1-AP, Proteintech, Philadelphia, PA, USA, 1:1000 for IB); cathepsin B (CTSB) (31718, CST, Danvers, MA, USA, 1:1000 for IB); LAMP1 (ab24170, abcam, Cambridge, UK, 1:1000 for IB and 1:200 for IF); collagen I (Col-1) (WL0088, Wanleibio, Liaoning, China, 1:500 for IB and abs131984, Absin Bioscience, Shanghai, China, 1:100 for IHC); fibronectin (Fn) (WL03180, Wanleibio, Liaoning, China, 1:500 for IB and NBP1-91258ss, Novus biological, Littleton, CO, USA, 1:200 for IHC); Beclin 1 (3738S, CST, Danvers, MA, USA, 1:1000 for IB); p62 (ab109012, abcam, Cambridge, UK, 1:10000 for IB); peroxidase-conjugated goat anti-rabbit IgG (H+L) (ZB2301, ZSGB-BIO, Beijing, China, 1:5000 for IB); rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H+L) (ZB0316, ZSGB-BIO, Beijing, China, 1:200 for IF); fluorescein-conjugated goat anti-rabbit IgG (H+L)(ZF0311, ZSGB-BIO, Beijing, China, 1:200 for IF); rhodamine (TRITC)-conjugated goat anti-mouse IgG (H+L) (ZF-0313, ZSGB-BIO, Beijing, China, 1:200 for IF); and Alexa Fluor ® 488 AffiniPure Fab Fragment Goat Anti-rabbit IgG (H+L) (111-547-003, Jackson ImmunoResearch, West Grove, PA, USA, 1:100 for IF).

Techniques: Activation Assay, In Vitro, Western Blot, Control, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Immunofluorescence, Ubiquitin Proteomics