Journal: bioRxiv

Article Title: Cyanine dye conjugates of a 2’-deoxycytidine-based auto- and mitophagy activator extend Caenorhabditis elegan s lifespan

doi: 10.64898/2026.01.23.701232

Figure Lengend Snippet: Results of primary screening of autophagy inducers among the reported compounds. A. Changes in the RFP/GFP ratio in the SH-SY5Y pMXs GFP-LC3-RFP cell line when treated with compounds under study. The results of ANOVA tests are indicated on the graph: * – p-value <0.05, ** – p-value <0.01, **** – p-value <0.0001. B. Dependence of the autophagy activation rate on selected substituents in four subgroups of compounds under study.

Article Snippet: Lentivirus was produced by transfecting HEK293T cells with either pMXs GFP-LC3-RFP (Addgene #117413, [ ]) or TOM20MTS-mCherry-EGFP-Tet-On (Addgene #109016, [ ]), together with the packaging plasmids pCMV-VSV-G (Addgene #8454) and psPAX2 (Addgene #12260), using Lipofectamine 3000 (Thermo Fisher Scientific, USA).

Techniques: Activation Assay

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: 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