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: Cell Death Discovery

Article Title: Linc00707 regulates autophagy and promotes the progression of triple negative breast cancer by activation of PI3K/AKT/mTOR pathway

doi: 10.1038/s41420-024-01906-7

Figure Lengend Snippet: A The MDA-MB-231 and MDA-MB-468 cell lines stably expressing stubRFP-sensGFP-LC3 were transfected with sh-con and sh-Linc00707 plasmids were observed by the fluorescence microscope, and the StubRFP-SensGFP-LC3 fluorescence spots were photographed under laser confocal microscopy. GFP represented autophagosomes, RFP represented the total number of autophagosomes and autolysosomes, yellow puncta in the merged image represented the flow rate of autophagic flow from autophagosomes to autolysosomes. Scale bar: 50 μm. B The changes of mitochondrial membrane potential of sh-con and sh-linc00707 groups in MDA-MB-231 and MDA-MB-468 were monitored by JC-1. Green fluorescence: JC-1 monomer, red fluorescence: JC-1 aggregates. Scale bar: 50 μm. C Western Blot analysis of the ratio of LC3-II/LC3-I and protein levels of SQSTM1/P62 in MDA-MB-231 and MDA-MB-468 cells transfected with sh-con or sh-Linc00707, oe-NC or oe-Linc00707. Western blot results are expressed as fold changes in relative band densities to control from three independent experiments. D Western Blot analysis of protein levels of p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR in MDA-MB-231 and MDA-MB-468 cells transfected with sh-con or sh-Linc00707, oe-NC or oe-Linc00707. Western blot results are expressed as fold changes in relative band densities to control from three independent experiments. E Western Blot analysis of protein levels of p-p70s6k, p70s6k, p-4EBP1, 4EBP1 in MDA-MB-231 and MDA-MB-468 cells transfected with sh-con or sh-Linc00707, oe-NC or oe-Linc00707. Western blot results are expressed as fold changes in relative band densities to control from three independent experiments. All experiments were repeated independently three times. Data are presented as means ± standard deviation. * P < 0.05.

Article Snippet: The antibodies specific to human proteins LC3 (1:1000) (bs-24359R, Bioss), anti-SQSTM1/P62 (1:1000) (bs-24359R, Bioss) were purchased from Bioss Biotech (Bioss Biotechnology Co. Ltd. Beijing, China).

Techniques: Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Confocal Microscopy, Membrane, Western Blot, Control, Standard Deviation

Journal: Cell Death Discovery

Article Title: Linc00707 regulates autophagy and promotes the progression of triple negative breast cancer by activation of PI3K/AKT/mTOR pathway

doi: 10.1038/s41420-024-01906-7

Figure Lengend Snippet: A Binding sites of miR-423-5p and MARCH2. B The relative luciferase activities in HEK293T cells co-transfected with MARCH2-WT or MARCH2-MUT and miR-423 overexpression vector (miR-423 mimic) or miR-NC. C Western blot analysis of the protein expression level of MARCH2 in MDA-MB-231 and MDA-MB-468 cells. Western blot results are expressed as fold changes in relative band densities to control from three independent experiments. D Western blot analysis of the protein expression level of MARCH2, LC3-II/LC3-I, SQSTM1/P62,p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR in MDA-MB-231 and MDA-MB-468 cells with different cotransfection. Western blot results are expressed as fold changes in relative band densities to control from three independent experiments. E Schematic representation of the mechanism showed that Linc00707/miR-423-5p/MARCH2 regulates autophagy and progression of TNBC cells via the PI3K/AKT/mTOR pathway. All experiments were repeated independently three times. Data are presented as means ± standard deviation. ns P ≥ 0.05, **P < 0.01.

Article Snippet: The antibodies specific to human proteins LC3 (1:1000) (bs-24359R, Bioss), anti-SQSTM1/P62 (1:1000) (bs-24359R, Bioss) were purchased from Bioss Biotech (Bioss Biotechnology Co. Ltd. Beijing, China).

Techniques: Binding Assay, Luciferase, Transfection, Over Expression, Plasmid Preparation, Western Blot, Expressing, Control, Cotransfection, Standard Deviation

Journal: Mediators of Inflammation

Article Title: Inhibitory Effect of Jinwujiangu Prescription on Peripheral Blood Osteoclasts in Patients with Rheumatoid Arthritis and the Relevant Molecular Mechanism

doi: 10.1155/2023/4814412

Figure Lengend Snippet: The percentage of MDC single standard (%) in each group. B1-L: negative areas represented the percentage of the cells without LC3 expression; B1-R: the positive areas represented the percentage of the cells expressing LC3. In comparison to the blank group, the value of ∗ P < 0.05 indicated a significant difference; the value of ∗∗ P < 0.01 indicated a highly significant difference.

Article Snippet: A total of 1 × 10 5 resuspended cells were centrifuged at 1000 RPM for 5 min, then the supernatant was discarded, and 5 μ L anti-LC3/FITC (Bioss, China, BS-8878R-FITC) was added, and the cells were gently resuspended.

Techniques: Expressing, Comparison

Journal: Frontiers in Pharmacology

Article Title: Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson’s Disease Model

doi: 10.3389/fphar.2020.618787

Figure Lengend Snippet: BBR mitigates autophagic impairment in MPTP-induced mice. (A) The representative immunohistochemical staining for MAP1LC3B in SN. (B) The number of MAP1LC3B positive cells in SN. Representative western blot bands (C) and the statistical graph (D) of MAP1LC3B and BECN1 in SN. (E) Transmission electron microscopy shown with autophagosomes (yellow arrows) in the SN. Data were expressed as the mean ± SD ( n = 6). * p < 0.05 compared with control group, # p < 0.05 compared with MPTP group, + p < 0.05 compared with MPTP + BBR group. MAP1LC3B, microtubule associated protein 1 light chain 3 beta; SN, substantia nigra; BECN1, beclin 1.

Article Snippet: The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026).

Techniques: Immunohistochemical staining, Staining, Western Blot, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson’s Disease Model

doi: 10.3389/fphar.2020.618787

Figure Lengend Snippet: BBR enhances autophagic activity in MPP + -treated BV2 cells. The representative immunofluorescent staining (A) and puncta (B) of MAP1LC3B in BV2 cells treated with MPP + at 200 μM and BBR at the concentration of 12.5, 25, and 50 μM (20 cells were analyzed per group for MAP1LC3B puncta counting). Representative western blots (C) and the statistical graph (D) of MAP1LC3B and BECN1 in BV2 cells treated with MPP + at 200 μM and BBR at the concentration of 12.5, 25, and 50 μM. The representative double-immunofluorescent staining (E) and puncta (E) of MAP1LC3B in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM (20 cells were analyzed per group for MAP1LC3B puncta counting). The representative monodansylcadaverine staining (G) and statistical graph (H) of autophagic vesicles in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM. Representative western blot bands (I) and the statistical graph (J) of MAP1LC3B and BECN1 in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM. Data were expressed as the mean ± SD ( n = 3). * p < 0.05 compared with untreated group, # p < 0.05 compared with MPP + group, + p < 0.05 compared with MPP + + BBR group. MAP1LC3B, microtubule associated protein 1 light chain 3 beta; BECN1, beclin 1.

Article Snippet: The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026).

Techniques: Activity Assay, Staining, Concentration Assay, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Slit2/Robo1 Mitigates DSS-induced Ulcerative Colitis by Activating Autophagy in Intestinal Stem Cell

doi: 10.7150/ijbs.42331

Figure Lengend Snippet: Expression of the autophagy proteins in DSS-induced mice. The coexpression of Lgr5 + colonic stem cells and the autophagy protein LC3 in (A) WT Slit and Slit2-Tg mice and (B) WT Robo1/2 and Robo1/2 +/- mice. Lgr5 (green) and LC3 (red) expression in the colonic epithelium. Sections were counterstained with DAPI (blue). (n=5 in each group; scale bar = 50 μm); (C) the photograph of the crypt isolated from colon tissue (scale bar= 200μm); (D) protein expression of LC3II/I in (D) WT Slit and Slit2-Tg mice and (F) WT Robo1/2 and Robo1/2 +/- mice (n=4); The expression of P62 in (E) WT Slit and Slit2-Tg mice and (G) WT Robo1/2 and Robo1/2 +/- mice (n=3-4 in each group). Detection of β-actin served as a loading control. Quantification of bands is expressed as density ratio of indicated protein/β-actin (A.U.); data are present as means ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Rabbit anti-mouse LC3 polyclonal antibody (1:500, ABclonal), rabbit anti-mouse p62 polyclonal antibody (1:500, Affinity Biosciences), and mouse anti-mouse β-actin (1:10000, Boster) were added and incubated at 4°C overnight.

Techniques: Expressing, Isolation, Control