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

Journal: bioRxiv

Article Title: RAB5c controls the assembly of non-canonical autophagy machinery to promote phagosome maturation and microbicidal function of macrophages

doi: 10.1101/2025.03.25.645097

Figure Lengend Snippet: (A) Analysis of time-lapse confocal microscopy of mCherry-RAB5c (mCh-RAB5c) at phagosomes containing opsonized-zymosan (Op-Zym) in RAW264.7 cells. Fluorescence profile was normalized by the MFI at 0” for each phagosome. Squares are the mean and shaded area is ± S.E.M. for 12 phagosomes (relative to Video S1) . (B) Representative confocal images of RAW264.7-mCh-RAB5c cells stimulated for 5 min with Op-Zym, Beads adsorbed with IgG (IgG-Beads), or albumin (BSA-Beads). Insets show mCh-RAB5c fluorescence intensity using the RAINBOW LUT. *, phagosomes. N: nuclei. Scale bar: 5 μm. (C-D) Representative confocal images (C) and MFI quantification (D) of mCh-LC3B at phagosomes in RAW246.7 cells transduced with scrambled shRNA (sh_Ctrl) or indicated shRNAs (#1; #2) against Rab5a, Rab5b, Rab5c, and fed Op-Zym for 25 min. Scale bar: 5 μm. (E-G) Representative confocal images (E) and MFI quantification of LC3 immunolabeling (AF-647) at phagosomes in RAW264.7 cells (F) and BMDM (G) transduced with sh_Ctrl or sh_ Rab5c and fed Op-Zym for 25 min. Blue: nuclei (Hoechst). Scale bar: 5 μm. (H) Immunoblot analysis of LC3, RAB5a/b/c and EEA1 in IgG-Bead- or BSA-Bead-containing phagosomes purified from RAW264.7 cells expressing sh_Ctrl or sh_ Rab5c 40 min after stimulation. UNC93B1 was used as loading control and GM130, constitutive of Golgi apparatus, as control of purification. Data are representative of 4-5 experiments performed independently. (I-J) Representative confocal images (I) and MFI quantification (J) of LAMP1 immunolabeling (AF-488) at phagosomes in RAW264.7 cells fed Op-Zym for 25 min. The fluorescence intensity is shown using the FIRE LUT. Green *: phagosome. Scale bar: 10 μm. (K-M) Flow cytometric analysis of Op-pHrodo Red-Zym in RAW264.7 cells 45 min post-stimulation. Histogram (K), quantification of MFI (L), and percentage of positive cells (M) for each group are shown. The black line on the histogram represents the fluorescence of non-stimulated sh_Ctrl cells. Bars are the mean value of biological replicates (n=4, each indicated as a circle), and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t -test. Data shown is representative of 3 experiments performed independently. (D, F, G, J) Gray objects are the MIF of each analyzed phagosome. Colored objects are the mean value for each independent experiment (n=3) and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t -test between the indicated groups.

Article Snippet: Anti-RAB5c [Thermo Fisher Scientific, PA5-39408; IB, 1:2,000; Immunofluorescence (IF), 1:300], anti-GAPDH [Cell Signalling Technology (CST), Clone D16H11, 5174T, IB, 1:10,000], anti-β-actin (CST, Clone 8H10D10, 12262, IB, 1:5,000), anti-Microtubule-associated protein 1 light chain B (anti-MAPLC3B/LC3B; CST, 27759, IB, 1:1,000), anti-LC3 (LC3A/LC3B/LC3C; MBL Int., PM036, IF: 1:250), anti-RAB5a (CST, 2143, IB, 1:1,000), anti-RAB5b (Santa Cruz, Clone F-9, sc373725, IB, 1:1,000), anti-EEA1 (CST, Clone C45B10, 3288, IB, 1:1,000), anti-UNC93B (Abcam, 69497, IB, 1:3,000), anti-GM130 (MBL, Clone 5G8, M1793, IB, 1:500), anti-UVRAG (CST, Clone D2Q1Z, 13115, IB, 1:1,000), anti-Phosphatidylinositol 3-kinase catalytic subunit type 3 (anti-PIK3C3/VPS34; Clone D9A5, CST 4263, IB, 1:1,000), anti-Beclin1 (CST, 3738, IB, 1:1,000), anti-Rubicon (Clone D9F7, CST, 8465, IB, 1:1,000), anti-p-p47 phox (S328) (Abcam, 111855, IF, 1:50), anti-p47 phox (Abcam, Clone erp27205, 308256, IB, 1:1,000; IF, 1:250), anti-p-p40 phox (Thr514) (CST, 4311, IB, 1:1,000), anti-p22 phox (Abcam, 75941 IB, 1:1,000), anti-gp91 phox (BD Bioscience, Clone53, 611414, IB, 1:2,000), anti-ERK1/2 (CST, Clone 1375F, 4695, IF, 1:500), anti-p-ERK1/2(Thr202/204) (CST, Clone 197G2, 9106, IF, 1:100), anti-ATP6V0d1 (Abcam, Clone erp18320-38, IB, 1:1,000), anti-ATP6V1A (Abcam, Clone erp19270, 199326, IB, 1:4,000; IF, 1:250), anti-ATG16L1 (MBL, Clone 1F12, M150-3, IB, 1:1,000), anti-ATG5/12 (CST, Clone D5F5U, 12994, IB, 1:1,000), anti-LAMP1 (CST, Clone E5N9Z, #99437, IF, 1:150), Alexa Fluor 647 polyclonal goat anti-rabbit IgG (Thermo Fisher Scientific, A21244, IF: 1:750), Alexa Fluor 488 polyclonal donkey anti-mouse IgG (Thermo Fisher Scientific, A21202, IF: 1:750), Alexa Fluor 647 polyclonal donkey anti-mouse IgG (Thermo Fisher Scientific, A31571, IF: 1:750), HRP-conjugated anti-rabbit IgG (CST, 7074, IB: 1:5,000), and HRP-conjugated anti-mouse IgG (CST, 7076, IB: 1:5,000) were used as indicated in manufactures′ datasheets.

Techniques: Confocal Microscopy, Fluorescence, Transduction, shRNA, Immunolabeling, Western Blot, Purification, Expressing, Control