Journal: Frontiers in Endocrinology

Article Title: Spatio-temporal dynamics of autophagy-associated genes in macrophage-driven atherosclerosis: an integrated omics and experimental study

doi: 10.3389/fendo.2026.1764263

Figure Lengend Snippet: Expression detection of key genes in AS macrophages. (A) statistical graph of SNX5, SMG1, GSK3A mRNA expression level. (B) The SNX5, SMG1, GSK3A protein expression levels: Left, typical western blots, Right, statistical graph. (C) Protein expression of autophagy markers LC3 and p62. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Raw264.7.

Article Snippet: In addition, the protein expression levels of LC3 (Affinity, AF7001) and P62 (BIOSS, bs-8878R) were assessed to evaluate autophagic flux, with β-actin (Proteintech, 66009-1-Ig) serving as the loading control for normalization.

Techniques: Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

doi: 10.3389/fimmu.2018.00211

Figure Lengend Snippet: Figure 2 | Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) suppressed autophagy in macrophages. (A–C) THP-1 macrophages were treated with recombinant human BD2 or BD3 or both peptides (1 µg/ml) for 6 h, and infected with Pseudomonas aeruginosa (PA) at multiplicity of infection 1 for 6 h. (D–G) RAW264.7 cells were transiently transfected with siBD2, siBD3, or both vs siNC for 24 h, and then infected with PA. (A,F) Cells were fixed, stained with anti- microtubule associated protein 1 light chain 3 (LC3) Alexa Fluro 488 fluorescent Ab, and then detected by immune fluorescence microscopy. Arrows indicate the LC3 puncta in THP-1 macrophages (A) and RAW264.7 cells (F). (B,G) Quantification of cells containing LC3 puncta in THP-1 macrophages (B) and RAW264.7 cells (G). (C,E) Protein levels of LC3-II were tested by western blot in THP-1 macrophages (C) and RAW264.7 cells (E). (D) The knockdown efficiency was tested by RT PCR. Data are shown as the mean ± SEM of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Primary antibody against microtubule associated protein 1 light chain 3 (LC3) (REF NB910-40435, Novus Biologicals, Littleton, CO, USA), anti-β-actin (REF A1978, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA), anti-Lamin B (REF sc-6216, clone B-10, Santa Cruz Biotechnology, CA, USA), anti-human betaDefensin 3 (REF AE1050, clone L3-18b-E1, Immundiagnostik), anti-human beta-Defensin 2 (REF AE1110, clone L12-4C-C2, Immundiagnostik, Produktion, Germany), anti-c-FOS (REF sc-52, clone E-4, Santa Cruz Biotechnology, CA, USA), and antiEGR1 (REF sc-189, clone C-19, Santa Cruz Biotechnology, CA, USA) were used in this study.

Techniques: Recombinant, Infection, Transfection, Staining, Fluorescence, Microscopy, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in immunology

Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

doi: 10.3389/fimmu.2018.00211

Figure Lengend Snippet: Figure 5 | Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) promoted macrophage-mediated phagocytosis and intracellular killing of Pseudomonas aeruginosa (PA) by inhibiting autophagy. RAW264.7 cells were transfected with siBD2, siBD3, siBecin1, or siATG7 for 24 h, and infected with PA at multiplicity of infection 25 for 1 or 2 h. Microtubule-associated protein 1 light chain 3 (LC3)-II protein expression was determined by western blot (A,D). Phagocytosis (B,E) and intracellular killing (C,F) was accessed by plate count assay. Data are shown as the mean ± SEM of three independent experiments (**p < 0.01; ***p < 0.001).

Article Snippet: Primary antibody against microtubule associated protein 1 light chain 3 (LC3) (REF NB910-40435, Novus Biologicals, Littleton, CO, USA), anti-β-actin (REF A1978, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA), anti-Lamin B (REF sc-6216, clone B-10, Santa Cruz Biotechnology, CA, USA), anti-human betaDefensin 3 (REF AE1050, clone L3-18b-E1, Immundiagnostik), anti-human beta-Defensin 2 (REF AE1110, clone L12-4C-C2, Immundiagnostik, Produktion, Germany), anti-c-FOS (REF sc-52, clone E-4, Santa Cruz Biotechnology, CA, USA), and antiEGR1 (REF sc-189, clone C-19, Santa Cruz Biotechnology, CA, USA) were used in this study.

Techniques: Transfection, Infection, Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

doi: 10.3389/fimmu.2018.00211

Figure Lengend Snippet: Figure 7 | Early growth response gene-1 (EGR1) or c-FOS enhanced autophagy in macrophages. (A–H) THP-1 macrophages were transfected with specific siRNA or overexpression plasmid for EGR1 or c-FOS for 24 h, and then infected with Pseudomonas aeruginosa (PA) for 1 h. Knockdown and overexpression effects of EGR1 (A,E) and c-FOS (B,F) were determined by western blot. Protein levels of microtubule associated protein 1 light chain 3 (LC3)-II (C,D,G,H) were tested by western blot in THP-1 macrophages before or after PA infection.

Article Snippet: Primary antibody against microtubule associated protein 1 light chain 3 (LC3) (REF NB910-40435, Novus Biologicals, Littleton, CO, USA), anti-β-actin (REF A1978, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA), anti-Lamin B (REF sc-6216, clone B-10, Santa Cruz Biotechnology, CA, USA), anti-human betaDefensin 3 (REF AE1050, clone L3-18b-E1, Immundiagnostik), anti-human beta-Defensin 2 (REF AE1110, clone L12-4C-C2, Immundiagnostik, Produktion, Germany), anti-c-FOS (REF sc-52, clone E-4, Santa Cruz Biotechnology, CA, USA), and antiEGR1 (REF sc-189, clone C-19, Santa Cruz Biotechnology, CA, USA) were used in this study.

Techniques: Transfection, Over Expression, Plasmid Preparation, Infection, Knockdown, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS, but not autophagy

doi: 10.1111/jcmm.14898

Figure Lengend Snippet: Anticancer drugs induced autophagy in lung cancer cells. A, A549 cells (2 × 10 5 ) were treated with the chemotherapeutic drugs cisplatin (2 μg/mL), LBH589 (100 nmol/L), or rapamycin (100 nmol/L), or a combination of two drugs for 24 h. The mRNA levels of ATG5 , ATG7 and BECN1 were assayed by RT‐qPCR. B, chemotherapeutic drug‐induced cells were pre‐treated with or without Bafilomycin A1 (BafA1, 20 nmol/L ) for 1 h followed by Western blot using anti‐LC3 antibody. C, A549 cells were treated with chemotherapeutic drugs for 48 h after transfection with pEGFP‐LC3 plasmid. LC3 punctate‐positive cells were observed by fluorescence microscopy and quantitation of the percentage of cells with punctate GFP‐LC3 fluorescence per total GFP‐LC3‐positive cells. Data represent mean ± SD calculated from three experiments of 100 transfected cells each. D, chemotherapeutic drug‐treated cells were stained with AO (1 μg/mL) for 20 min. Autophagic cells were analysed using flow cytometry. Results are representative of at least three independent experiments. * P < .05 and ** P < .01 compared with untreated cells

Article Snippet: Rapamycin, LC3 antibody and BNIP3 antibody were obtained from Calbiochem, Novus Biologicals and Abcam, respectively.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Quantitation Assay, Staining, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS, but not autophagy

doi: 10.1111/jcmm.14898

Figure Lengend Snippet: Autophagic inhibitor 3‐MA suppressed chemotherapeutic drug‐induced autophagy. A, A549 cells (2 × 10 5 ) were treated with cisplatin (2 μg/mL), LBH589 (100 nmol/L), or rapamycin (100 nmol/L), or a combination of two drugs for 48 h. Cells were treated with or without 3‐MA (3 mmol/L) 1 h prior to analysis. Western blot was conducted using an anti‐LC3 antibody. B, C, pEGFP‐LC3‐transfected A549 cells were treated with chemotherapeutic drugs with or without 3‐MA 1 h prior to analysis. B, Quantitation of the number of GFP‐LC3 puncta per cell was conducted using MetaMorph software. C, LC3 punctate‐positive cells were quantitated by cells with punctate GFP‐LC3 fluorescence per total GFP‐LC3‐positive cells. D, A549 cells were treated with chemotherapeutic drugs with or without 3‐MA 1 h prior to analysis, followed by staining with AO (1 μg/mL). Autophagic cells were analysed using flow cytometry. Results are representative of at least three independent experiments. * P < .05 and ** P < .01

Article Snippet: Rapamycin, LC3 antibody and BNIP3 antibody were obtained from Calbiochem, Novus Biologicals and Abcam, respectively.

Techniques: Western Blot, Transfection, Quantitation Assay, Software, Fluorescence, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) GO analysis revealed that cardiomyocytes under HHCY conditions were mainly enriched in mitochondrial processes, mitochondrial function, and inner mitochondrial membrane signaling pathways. (B) GSEA of AP39-intervened samples that showed significant enrichment and upregulation of the mitophagy pathway. (C) Cystoscope analysis the top 5 ranked genes were SQSTM1, PINK1, HIF1α, FUNDC1, and TBK1. Expression of FUNDC1, LC3A/B in SD rats induced by HHCY (D) and H9c2 cardiomyocytes (E) in each group. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; $p < 0.05 vs. HHCY + AP39, $$$p < 0.005 vs. HHCY + AP39); (F) Expression of P53,P16 in H9c2 cardiomyocytes in each group after siRNA interference. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; $$$p < 0.005 vs. HHCY + AP39); (G) Observation of changes in Mitophagy formation in each group under transmission electron microscopy. Expression of MFN1, MFN2, Drp1 in SD rats induced by HHCY (H) and H9c2 cardiomyocytes (I) in each group. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; ***p < 0.005 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; ###p < 0.005 vs. HHCY; $p < 0.05 vs. HHCY + AP39, $$p < 0.01 vs. HHCY + AP39, $$$p < 0.005 vs. HHCY + AP39); (J) Co-localization of FUNDC1 and LC3B in cardiomyocytes in each group under fluorescence microscopy. (K) Mitochondrial membrane potential changes after siRNA interference. (Red fluorescence indicates high mitochondrial membrane potential; green fluorescence indicates low mitochondrial membrane potential). Gene Ontology: GO; high homocysteine: HHCY; Gene Set Enrichment Analysis: GSEA.

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Membrane, Protein-Protein interactions, Expressing, Control, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) Co-Immunoprecipitation (Co-IP) analysis showing the binding of FUNDC1 to DRP1 in cardiomyocytes of each group; (B) Expression of P53, P16 in H9c2 cardiomyocytes in each group after siRNA interference. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; $p < 0.05 vs. HHCY + AP39); (C) Detection of cellular senescence in heart tissues of each group (upper panel) and in cardiomyocytes of each group (lower panel) by β-galactosidase assay.

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) KEGG analysis revealed that cardiomyocytes under HHCY conditions were mainly enriched in ubiquitination pathway. (B) Heatmap showing differentially expressed CULLIN family genes in HHCY hearts. P value is shown. (C) Expression of NEDD8, Cul4b in SD rats induced by HHCY in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY); (D) Co-Immunoprecipitation (Co-IP) analysis showing the binding of FUNDC1 to Cul4b in cardiomyocytes of each group; (E–G) Expression of Cul4b, FUNDC1, P53, P16 in H9c2 cardiomyocytes in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY); (H) Co-immunoprecipitation (Co - IP) and ubiquitination assays were performed to assess FUNDC1 ubiquitination in Control, HHCY, and HHCY + AP39 groups. (I,J) Expression of FUNDC1, NEDD8, CUL48in H9c2 cardiomyocytes in each group. (K) Expression of NEDD8, CUL48, S-NEDD8, S-CUL4B in H9c2 cardiomyocytes in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; $p < 0.05 vs. HHCY + AP39).

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Ubiquitin Proteomics, Expressing, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay

Journal: Inflammatory Bowel Diseases

Article Title: Microbial Disruption of Autophagy Alters Expression of the RISC Component AGO2, a Critical Regulator of the miRNA Silencing Pathway

doi: 10.1097/mib.0000000000000553

Figure Lengend Snippet: FIGURE 2. Disruption of autophagy increases AGO2 in MDAMC cells. A, Western blotting for AGO2 and LC3 in lysates obtained from MDAMC cells treated with control siRNA, ATG12 siRNA, or VacA. B, Graph depicts quantification of AGO2 relative to b-actin levels (n ¼ 3 experiments **P , 0.01, analysis of variance with Dunnett’s post hoc test). C, Representative confocal image of LC3-GFP expressing control siRNA or ATG12 siRNA cells, in the presence or absence of VacA. LC3 is depicted in green, AGO2 is depicted in red, and p62 is depicted in blue.

Article Snippet: Proteins were then transferred onto a nitrocellulose membrane that was blocked with 5% milk in Tris-buffered saline with tween and probed with antibodies diluted in 5% milk in Tris-buffered saline with tween: 1:500 AGO2 rabbit polyclonal antibody (Abcam), 1:2000 LC3 rabbit polyclonal antibody (Novus Biologicals, Oakville, Canada), or 1:1000 GFP rabbit polyclonal antibody (Invitrogen, Life Technologies) and developed with the appropriate horseradish peroxidase– conjugated secondary antibody.

Techniques: Disruption, Western Blot, Control, Expressing

Journal: Inflammatory Bowel Diseases

Article Title: Microbial Disruption of Autophagy Alters Expression of the RISC Component AGO2, a Critical Regulator of the miRNA Silencing Pathway

doi: 10.1097/mib.0000000000000553

Figure Lengend Snippet: FIGURE 1. Autophagy-deficient MEF cells display increased AGO2. A, Representative Western blot for AGO2 and LC3 in lysates obtained from Atg52/2 or WT cells. B, Representative confocal image of ATG52/2 or WT LC3-GFP expressing cells. LC3 is depicted in green. AGO2 is depicted in red. C, Graph depicts quantification of AGO2 relative to b-actin levels (n ¼ 5 experiments; *P , 0.05). D, Representative Western blot for AGO2 and LC3 in lysates obtained from Atg52/2 or WT cells treated with bafilomycin to disrupt autophagy.

Article Snippet: Proteins were then transferred onto a nitrocellulose membrane that was blocked with 5% milk in Tris-buffered saline with tween and probed with antibodies diluted in 5% milk in Tris-buffered saline with tween: 1:500 AGO2 rabbit polyclonal antibody (Abcam), 1:2000 LC3 rabbit polyclonal antibody (Novus Biologicals, Oakville, Canada), or 1:1000 GFP rabbit polyclonal antibody (Invitrogen, Life Technologies) and developed with the appropriate horseradish peroxidase– conjugated secondary antibody.

Techniques: Western Blot, Expressing

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Cosmo Bio anti-LC3B specifically recognizes LC3B in EGFP-LC3B transfected cells. (A) Schematic overview of endo-lysosomal and autophagic compartments, outlining their characteristic morphological features and presence of LC3. (B-C) EGFP-LC3B transfected cells were fixed with 2% PFA+0.2% GA for 3 h and double labeled for LC3B and GFP and protein A gold (PAG). (B) The labeling intensities of anti-GFP (1:400; PAG15) and Cosmo Bio anti-LC3B (1:10; PAG10) correlate in cells with high and low EGFP-LC3B overexpression. (C) Example of double labeling with anti-GFP (1:400; PAG15) and another LC3 antibody (CST, 4108, 1:15; PAG10) showing only PAG15 on EGFP-LC3B overexpressing cells. AL, autolysosome; M, mitochondrion; PM, plasma membrane. Scale bars: 300 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Transfection, Labeling, Over Expression, Clinical Proteomics, Membrane

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Performance of commercial LC3 antibodies in IF and immuno-EM.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques:

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Imaging autophagy by conventional EM and immunofluorescence. (A) Conventional EM of control U2OS cells. Endo-lysosomal compartments are identified by morphology. (B) Conventional EM of U2OS cells starved for 2.5 h in the presence of BafA1 showing an accumulation of different types of autophagic compartments. Autophagosomes (AP) are recognized by their double-membrane that encapsulates part of the cytoplasm with the same density as the surrounding cytoplasm and containing multiple membrane structures. Autolysosomes (AL) are vacuoles lined by a single membrane. Their lumen is heterogenous in content (membranes, vesicles, amorphous material) and electron density (from light to dense). Both AP and AL contain recognizable endoplasmic reticulum (ER) cisternae (arrows). Asterisks indicate autophagic content. (C-E) IF of semi-thin cryosections of control and starved, BafA1-treated U2OS cells, fixed either with 4% PFA ON (PFA) or 2% PFA+0.2% GA for 2 h (PFA+GA) and labeled with Cosmo Bio anti-LC3B (1:10) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:300). Images are recorded with identical settings and represented with equal intensity ranges. PFA and PFA+GA fixed cells contain comparably intense LC3B puncta. AL, autolysosome; AP, autophagosome; E, endosome; Ly, lysosome; M, mitochondrion; PM, plasma membrane. Scale bars: 500 nm (A-B); 20 µm (C-E).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Imaging, Immunofluorescence, Control, Membrane, Labeling, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Correlative light electron microscopy (CLEM) of endogenous LC3B on ultrathin cryosections. (A) Ultrathin (60–70 nm) cryosection of starved, BafA1-treated U2OS cells (fixation 4% PFA ON) labeled for LC3B (1:10), rabbit anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG and PAG10, showing IF puncta (green) and nuclei (Hoechst, blue). (B) Low magnification EM picture of same section as in (A). Arrows point to LC3B-immunogold labeled compartments (gold not visible at this magnification). (C) Overlay of the fluorescent and EM images in (A) and (B). Higher magnifications of the boxed areas in (C) are shown in (D-F). (D) A phagophore (PG) is visible as a ring of LC3B-positive vesicles. Arrows indicate visible membrane contours. (E, F) Two examples of autolysosomes (AL). LC3B (PAG10) is predominantly associated with autophagic content located in the AL lumen (indicated by asterisks). Note that the higher magnifications in D-F are rotated relative to B. N, nucleus. Scale bars: 2 µm (A-C), 200 nm (D-F).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Electron Microscopy, Labeling, Membrane

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Effect of different fixation and labeling regimes on LC3 immuno-EM labeling efficiency. (A-D) Electron micrographs of U2OS cells starved in the presence of BafA1 for 2.5 h. Cells were fixed and immunolabeled for LC3B (1:10; PAG10) according to the standard or fast labeling protocol as indicated. (A, B) Cells fixed ON with 4% PFA labeled by the standard (A) or fast (B) protocol. Both conditions show abundant LC3B label on autophagic content (asterisks) present in autolysosomes (AL). (C) Cells fixed for 15 min with 4% PFA, followed by 6 days of 0.6% PFA and labeled by the fast protocol. (D) Cells fixed for 3 h with 2% PFA+0.2% GA labeled by the standard protocol. Note a reduction in LC3B label compared to A-C. (E) Quantification of the number of LC3B-representing PAG10 particles per organelle for the conditions shown in A-D. Cells were randomly screened for LC3B-positive organelles. The number of organelles (N) screened was 80, 71, 74 and 25 for conditions indicated in A, B, C and D, respectively. Only the PFA+GA condition significantly differs from the others at p ≤ 0.0001 using Student’s t -test assuming unequal variance (***). (F) Starved, BafA1-treated U2OS cells fixed with 4% PFA for 15 min followed by 6 days with 0.6% PFA. Representative image of an autophagosome (AP) labeled for LC3B (1:6) using a gold enhancement step after PAG5 labeling. AP, autophagosome; ER, endoplasmic reticulum; M, mitochondrion. Scale bars: 200 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Labeling, Immunolabeling

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Effect of different fixation and labeling regimes on morphology. U2OS cells starved for 2.5 h in the presence of BafA1, fixed and labeled with Cosmo Bio anti-LC3B and PAG10 according to the indicated fixation and labeling protocols. ( A, E, G ) Typical examples of autophagosomes (AP) using different fixation and labeling regimes. The double membrane is partially extracted resulting in a halo with remnants of inner (open arrowheads) and outer (black arrowheads) membrane. Arrows indicate LC3 PAG10 associated with the outer membrane. (C) Phagophore (PG), recognizable by the edge (shafted arrowhead) of the cup-shaped double membrane. ( B, D, F, H ) Examples of autolysosomes (AL) using the indicated fixation and labeling regimes. Autolysosomes typically contain autophagic content (asterisks) positive for LC3B, are sometimes filled with internal vesicles and display an overall heterogeneous content. The different PFA fixations yield comparable morphologies. The PFA+GA fixation yields an overall better ultrastructure, yet still results in the halo around phagophores and autophagosomes. Dilutions Cosmo Bio LC3B antibody: 1:4 (A, C, D), 1:10 (B, E-H). Asterisk, autophagic content. ER, endoplasmic reticulum; G, Golgi; M, mitochondrion; N, nucleus; PM, plasma membrane. Scale bars: 100 nm (A, C, E, G, H), 200 nm (B, D, F).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Labeling, Membrane, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Immuno-EM of endogenous LC3B in primary cells and tissues. (A-B) Primary mouse macrophages were starved for 30 min without addition of BafA1. LC3B labeling (1:6; PAG10, arrows) is detected on autophagosomes (AP) and autolysosomes (AL). Gold particles are associated with the inner (open arrowheads) and outer (black arrowheads) autophagosome membrane. (A) Cells fixed ON with 4% FPA. (B) Cells fixed with 2% PFA+0.2% GA for 2 h. (C-H) In rat pancreas and liver (1:10; PAG10), in the absence of lysosomal inhibitors, LC3B labeling is detected mainly on autophagosomes (AP) and occasionally on an autolysosome (AL). Gold particles are associated with the inner and outer (arrows) autophagosome membrane. (C-F) Rat exocrine pancreas perfusion fixed with 2% PFA+0.2% GA. (C) Overview. (D, E) Examples of autophagosomes (AP) encapsulating mainly ER membranes. (F) Group of 2 (not-labeled) autolysosomes (AL) and an autophagosome (AP) near the Golgi (G), enlarged from the box in (C). LC3 immunogold label is present on inner and outer (arrows) AP membrane. (G, H) Rat liver perfusion fixed with 4% PFA. (H) LC3 immunogold label on a group of autophagosomes (AP) and an autolysosome (AL) in a hepatocyte, enlarged from the box in (G). Arrows indicate LC3 gold on the outer AP membrane. BC, bile canaliculus; EE, early endosome; ER, endoplasmic reticulum; M, mitochondrion; NE, nuclear envelope; PM, plasma membrane; SG, secretory granule. Scale bars: 100 nm (D), 200 nm (A, B, E, F, H), 1 μm (C), 500 nm (G).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Labeling, Membrane, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: LC3B colocalizes with endocytosed BSA 5 , LAMP1 and SQSTM1/p62 in autolysosomes of starved, BafA1-treated U2OS cells. (A) Timeline of experimental setup. (B) Epon section showing BSA 5 -containing autolysosome (AL) with similar morphological features as the AL in (C). (C) LC3B (1:4; PAG10) is present in an autolysosome (AL) also containing BSA 5 (black arrowheads). White arrowheads mark the limiting membrane of the AL, showing that LC3B and BSA 5 colocalize in the AL lumen. (D) Double labeling of LAMP1 (1:60; PAG10) and LC3B (1:10; PAG15). Accumulation of LC3B inside a LAMP1-positive autolysosome (AL) also positive for endocytosed BSA 5 (black arrowheads). (E) Colocalization of LC3B (1:6; PAG15) and SQSTM1/p62 (1:100; PAG10) in a typical autolysosome (AL). SQSTM1/p62 label (arrows) marks only a subset of the LC3B-positive material. Fixation for immuno-EM: (C) and (E) 4% PFA ON; (D) 2% PFA, 0.2% GA, 3 h. Asterisks, autophagic content. PM, plasma membrane. Scale bars: 200 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Membrane, Labeling, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: LC3B is incorporated in autolysosomes after extended incubation with BafA1. U2OS cells were treated with BafA1 for the indicated durations and starved in presence of BafA1 using EBSS for 2.5 h before fixation with 4% PFA. Double labeling of LC3B (1:10; PAG15) and LAMP1 (1:100; PAG10) reveals that in all conditions the majority of LC3B label is found in LAMP1-positive autolysosomes (AL). (A) 2.5 h; (B) 5 h; (C) 10 h; (D) 24 h of BafA1 incubation. (E) Quantification of >50 LC3B-positive organelles scored for presence of LAMP1 label per condition. These results indicate that fusion of LC3B-positive autophagosomes with lysosomes proceeds after BafA1 treatment. For more examples of LC3B labeling after BafA1 treatment and the effect on autolysosome morphology, see Fig. S3. Asterisks, autophagic content. M, mitochondrion; N, nucleus. Scale bars: 200 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Incubation, Labeling