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: 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 beta-Defensin 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 anti-EGR1 (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: 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 beta-Defensin 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 anti-EGR1 (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: 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 beta-Defensin 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 anti-EGR1 (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: Frontiers in pharmacology

Article Title: Shengma Biejia Decoction Inhibits Cell Growth in Multiple Myeloma by Inducing Autophagy-Mediated Apoptosis Through the ERK/mTOR Pathway.

doi: 10.3389/fphar.2021.585286

Figure Lengend Snippet: FIGURE 3 | SMBJD is safe and could inhibit the expression of autophagy-related proteins, promote apoptosis-related proteins in vivo. (A) Representative pictures of peer group mouse liver, spleen, and kidney after HE staining (× 200), scale bar 100 µm. (B) The expression levels of P62, LC3, and cleaved caspase-3 in transplanted tumors were detected using immunohistochemistry (× 200), scale bar 100 µm. (C) Extracts from transplanted tumors were analyzed for autophagy- related and apoptosis-related proteins expression by western blot. (D) The corresponding expression of P62, LC3, and cleaved caspase-3 levels in tumor tissue are shown as histograms. (E) The corresponding autophagy-related and apoptosis-related proteins expression by western blot are shown as histograms. (*p < 0.05 compared with the model group).

Article Snippet: The membranes were blocked with 5% fat-free milk with 0.1% Tween-20 in 0.02 ml of TBS buffer (TBST) for 1 h and incubated with primary antibodies against P62 (1:1,000–4,000, Proteintech, IL, United States), LC3 (1:600–2,500, Proteintech, IL, United States), Bax (1:1,000, CST, MA, United States), Bcl-2 (1:1,000, CST, MA, United States), caspase-3 (1:1,000, CST, MA, United States), cleaved caspase-3 (1:1,000, CST,MA, United States), ERK1/2 (1:10,000, Abcam, Cambridge, United Kingdom), phosphorylated ERK1/2 (1:5,000–10,000, Abcam, Cambridge, United Kingdom), Ser2448 mTOR (1:500–2,000, Sciben, Nanjing, China), and phosphorylated Ser2448 mTOR (1: 500–2,000, Sciben, Nanjing, China) at 4°C overnight.

Techniques: Expressing, In Vivo, Staining, Immunohistochemistry, Western Blot

Journal: Frontiers in pharmacology

Article Title: Shengma Biejia Decoction Inhibits Cell Growth in Multiple Myeloma by Inducing Autophagy-Mediated Apoptosis Through the ERK/mTOR Pathway.

doi: 10.3389/fphar.2021.585286

Figure Lengend Snippet: FIGURE 4 | SMBJD represses the growth, inhibits autophagy, and promotes apoptosis of H929 and U266 cells. (A) The cell viability of H929 and U266 cells after different concentrations and time of SMBJD treatment were analyzed with MTT assay. (B) IC50 value of H929 and U266 cells after SMBJD intervention. (C) H929 and U266 cells were transiently transfected with Ad-GFP-LC3 for 24 h. Then the cells were cultured with SMBJD for another 36 h. The formation of GFP-LC3 puncta were examined by a confocal microscope and typical images were presented (× 400), scale bar 2 µm. (D) Annexin V/PI staining of H929 and U266 cells apoptosis rate treated with SMBJD for 36 h (E, F) The quality graphs of C and D. Data are expressed as the means ± SD (nsP > 0.05 compared with each other, *p < 0.05 compared with control group).

Article Snippet: The membranes were blocked with 5% fat-free milk with 0.1% Tween-20 in 0.02 ml of TBS buffer (TBST) for 1 h and incubated with primary antibodies against P62 (1:1,000–4,000, Proteintech, IL, United States), LC3 (1:600–2,500, Proteintech, IL, United States), Bax (1:1,000, CST, MA, United States), Bcl-2 (1:1,000, CST, MA, United States), caspase-3 (1:1,000, CST, MA, United States), cleaved caspase-3 (1:1,000, CST,MA, United States), ERK1/2 (1:10,000, Abcam, Cambridge, United Kingdom), phosphorylated ERK1/2 (1:5,000–10,000, Abcam, Cambridge, United Kingdom), Ser2448 mTOR (1:500–2,000, Sciben, Nanjing, China), and phosphorylated Ser2448 mTOR (1: 500–2,000, Sciben, Nanjing, China) at 4°C overnight.

Techniques: MTT Assay, Transfection, Cell Culture, Microscopy, Staining, Control

Journal: Frontiers in pharmacology

Article Title: Shengma Biejia Decoction Inhibits Cell Growth in Multiple Myeloma by Inducing Autophagy-Mediated Apoptosis Through the ERK/mTOR Pathway.

doi: 10.3389/fphar.2021.585286

Figure Lengend Snippet: FIGURE 5 | SMBJD inhibits autophagy-related proteins and promotes apoptosis-related proteins of multiple myeloma cell lines H929 and U266 (A) Western blot analysis of autophagy-related and apoptosis-related proteins expression after SMBJD administration for 36 h in both cells. (B) Western blot analysis of LC3 expression after SMBJD, 3-MA, Baf-A1, and SMBJD + Baf-A1 administration for 36 h in H929 and U266 cells.

Article Snippet: The membranes were blocked with 5% fat-free milk with 0.1% Tween-20 in 0.02 ml of TBS buffer (TBST) for 1 h and incubated with primary antibodies against P62 (1:1,000–4,000, Proteintech, IL, United States), LC3 (1:600–2,500, Proteintech, IL, United States), Bax (1:1,000, CST, MA, United States), Bcl-2 (1:1,000, CST, MA, United States), caspase-3 (1:1,000, CST, MA, United States), cleaved caspase-3 (1:1,000, CST,MA, United States), ERK1/2 (1:10,000, Abcam, Cambridge, United Kingdom), phosphorylated ERK1/2 (1:5,000–10,000, Abcam, Cambridge, United Kingdom), Ser2448 mTOR (1:500–2,000, Sciben, Nanjing, China), and phosphorylated Ser2448 mTOR (1: 500–2,000, Sciben, Nanjing, China) at 4°C overnight.

Techniques: Western Blot, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Exosomes derived from fibroblasts in DFUs delay wound healing by delivering miR-93-5p to target macrophage ATG16L1.

doi: 10.1016/j.bbadis.2024.167640

Figure Lengend Snippet: Fig. 3. miR-93-5p affects macrophage autophagy function by affecting Atg16L1. (A) Nine mRNAs were identifed based based on TargetScan, miRDB, miRTarBase and Human Autophage Database. (B) Schematic representation of the wild-type and mutant-type binding site between the 3′UTR of ATG16L1 and miR-93-5p. (C) Relative luciferase activity of 3′UTR-ATG16L1-luc constructs in HEK293T cells after transfection of miR-93-5p mimics/NC; n = 3 per group. (D) The protein levels of ATG16L1 in bone marrow-derived macrophages transfected with miR-93-5p NC/mimics or PBS (Control) by Western blot. (E) The protein levels of ATG16L1 in skin tissues from normal or diabetic wound (DFUs) by Western blot. (F) Immunofluorescence staining for ATG16L1 (red) and F4/80 (green) in skin tissues from normal or diabetic wound (DFUs); scale bar, 100 μm. (G) The protein levels of LC3b and P62 in BMDMs transfected with miR-93-5p NC/mimics or PBS by Western blot, immunofluorescence staining for LC3b (green) in bone marrow-derived macrophages transfected with miR-93-5p NC/mimics or PBS; scale bar, 50 μm. (H) The protein levels of ATG16L1, LC3 and P62 in fibroblast-derived exosomes treated bone marrow-derived macrophages transfected with miR-93-5p inhibitor NC/in hibitor or PBS (Control) by Western blot. Data are expressed as mean ± SD, ns P > 0.05, **P < 0.01 (unpaired t-test or ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Next, sections were incubated overnight at 4 ◦C with primary antibodies against F4/80 (Servicebio, GB11027), Atg16L1 (CST, #8089), LC3B (Proteintech, 81004-1-RR), α-SMA (Proteintech,14395-1AP) and Collagen Type I (Proteintech, 14695-1-AP).

Techniques: Mutagenesis, Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Derivative Assay, Control, Western Blot, Immunofluorescence, Staining