Journal: Cell & Bioscience

Article Title: Ubiquitinated AIF is a major mediator of hypoxia-induced mitochondrial dysfunction and pulmonary artery smooth muscle cell proliferation

doi: 10.1186/s13578-022-00744-3

Figure Lengend Snippet: Effects of hypoxia and AIF on mitophagy and autophagy in PASMCs. A AIF influenced mitochondrial function by obstructing Tom20 and Lamp2a colocalization, as indicated by the decreased yellow area. Scale bars: 50 μm (n = 4). B PASMCs were exposed to hypoxia for 24 h, and the expression of Pink and Parkin was evaluated by Western blotting (n = 5). C AIF overexpression mitigated the alteration of mitophagy in PASMCs as determined by Mitophagy Keima-Red plasmid transfection. The number of mitochondrial autophagosomes (yellow dots) was calculated (n = 5). Scale bars: 50 μm. D The expression of LC3B-II, P62 and ATG5/7 was evaluated by Western blotting (n = 5). E Autophagic flux and the formation of autophagosomes were detected (n = 6). Scale bars: 50 μm. F Representative electron micrograph of cells after Nor and Hyp treatment. Asterisks represent autophagosomes and arrows represent autolysosomes. Scale bars: 500 nm (n = 3). All data are presented as the means ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; Nor normoxia, Hyp hypoxia, NC negative control

Article Snippet: Antibodies against Cyclin A, Cyclin D, PCNA, UB, P62, ATG5 and ATG7 (Catalog numbers BM1582, BM4272, BM0104, BM4359, BA2849, BA3525 and BA3527) were obtained from Boster (Wuhan, China).

Techniques: Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Standard Deviation, Negative Control

Journal: Oncology Letters

Article Title: Endocan silencing induces programmed cell death in hepatocarcinoma

doi: 10.3892/ol.2017.6857

Figure Lengend Snippet: The levels of LC3, ATG5, ATG7, DRAM and Beclin-1 protein expression were measured following transfection with endocan siRNA and normalized to β-actin. (A) Representative blot and (B) quantification of autophagy-associated protein expression. The expression levels of all the autophagy-associated proteins were significantly increased in endocan siRNA-treated cells compared with those in the NC and control groups. *P<0.05 in siRNA groups vs. NC groups. NC, negative control; siRNA, small interfering RNA; LC3, microtubule associated protein 1 light chain 3 α; ATG, autophagy related; DRAM, DNA damage regulated autophagy modulator 1.

Article Snippet: The primary antibodies were as follows: Endocan antibody (sc-515304; Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight; LC3 antibody (AL221; Beyotime Institute of Biotechnology; 1:500) at 4°C overnight; ATG5 antibody (PA2260; Boster Biological Technology, Pleasanton, CA, USA; 1:400,) at 4°C overnight; ATG7 antibody (PB9479; Boster Biological Technology; 1:400) at 4°C overnight; DRAM antibody (bs-4233R, BIOSS, Beijing, China; 1:500) at 4°C overnight; Beclin-1 antibody (AB123; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight and NF-κB p65 antibody (AF0246; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight.

Techniques: Expressing, Transfection, Control, Negative Control, Small Interfering RNA

Journal: mBio

Article Title: PP2C phosphatases Ptc1 and Ptc2 dephosphorylate PGK1 to regulate autophagy and aflatoxin synthesis in the pathogenic fungus Aspergillus flavus

doi: 10.1128/mbio.00977-23

Figure Lengend Snippet: Dephosphorylation of PGK1 S203 promotes autophagy in A. flavus. ( A ) Representative TEM images of autophagosomes in PGK1::eGFP, PGK1S203D::eGFP, and PGK1S203A::eGFP strains for 5 days. Red arrow, autophagic vesicles; scale bar: 500 nm. ( B ) Quantification of an average number of autophagic vesicles per cell. Error bars represent SD and statistical significance was determined by ANOVA (Dunnett t -test). ** presents P ≤ 0.01. ( C ) The ratio of free GFP to the total GFP signal in three independent experiments from PGK1::eGFP, PGK1 S203D ::eGFP, and PGK1 S203A ::eGFP, as described in (left panel). The β-actin served as loading control for standardization of each relevant sample. Error bars represents SD and statistical significance was determined by ANOVA (Dunnett t -test). ** present P ≤ 0.01. ( D ) PGK1::eGFP, PGK1S203D::eGFP, and PGK1S203A::eGFP were analyzed using immunoblotting. The relative expression levels of Atg5, Atg5-Atg12, and Atg8-PE/Atg8 were normalized by β-actin for each group. The intensity of fusion protein in WT was defined as 100. ( E ) Proposed regulation of aflatoxin production via Ptc1 and Ptc2 phosphatases. Ptc1 and Ptc2 are involved in aflatoxin biosynthesis and autophagy induction by affecting mitochondria-translocated PGK1. The phosphorylated S203 of PGK1 negatively regulates synthesis of acetyl-CoA in mitochondria and autophagy. Broken arrows represent inhibited directions or reactions. The red cross indicates deficiency.

Article Snippet: The following antibodies were used in this study: anti-GFP (1:5,000, 2057R, Dia-An), anti-GAPDH (1:5,000, 60004–1-Ig, Proteintech), anti-β-actin (1:5,000, PTM-5018, PTM Biolabs), anti-Slt2 with p44/42 MAPK (Erk1/2) (1:5,000, Cell Signaling Technology, 9102), anti-phospho-Slt2 with phospho-p44/42 MAPK (Erk1/2) antibody (1:2,000, Cell Signaling Technology, 4377), anti-ATG5 (1:1,000, PTM-5868, PTM Biolabs), anti-Atg8 (1:1,000, PTM-6384, PTM Biolabs), anti-PGK1 (1:2,000, A14039, ABclonal), anti-TIMM10 (1:2,000, A4626, ABclonal), anti-H3 (1:2,000, 68345–1-Ig, Proteintech), anti-pan-phospho-Ser (1:2,000, ICP9806, ImmuneChem), anti-pan-phospho-Thr (1:2,000, ICP9807, ImmuneChem), anti-phospho-PGK1 S203 (1:200, SAB487P, Signalway Antibody), and goat anti-rabbit IgG(H+L) (1:5,000, SA00001-2, Proteintech).

Techniques: De-Phosphorylation Assay, Western Blot, Expressing

Journal: Brain and Behavior

Article Title: Relationship of Autophagic Dysfunction With the Quality of Life and Sleep, Depression and Disease Severity in Patients With Restless Legs Syndrome

doi: 10.1002/brb3.70172

Figure Lengend Snippet: Comparison of Serum ATG3 (a) and ATG5 (b) expressions levels in patients with RLS. RLS: restless leg syndrome, ATG3: autophagy‐related gene 3, ATG5: autophagy‐related gene 5.

Article Snippet: The ATG5 measure has a sensitivity of 0.188 ng/mL (Finetest, EH1729).

Techniques: Comparison

Journal: Brain and Behavior

Article Title: Relationship of Autophagic Dysfunction With the Quality of Life and Sleep, Depression and Disease Severity in Patients With Restless Legs Syndrome

doi: 10.1002/brb3.70172

Figure Lengend Snippet: Relationship of ATG3 and ATG5 with quality of life and sleep, the depression and laboratory markers in patients with restless legs syndrome.

Article Snippet: The ATG5 measure has a sensitivity of 0.188 ng/mL (Finetest, EH1729).

Techniques:

Journal: Brain and Behavior

Article Title: Relationship of Autophagic Dysfunction With the Quality of Life and Sleep, Depression and Disease Severity in Patients With Restless Legs Syndrome

doi: 10.1002/brb3.70172

Figure Lengend Snippet: The evaluation of potential predictive values of ATG3 (a) and ATG5 (b) in patients with RLS using ROC curve analyses. RLS: restless leg syndrome, ATG3: autophagy‐related gene 3, ATG5: autophagy‐related gene 5, ROC: receiver‐operating characteristic curve.

Article Snippet: The ATG5 measure has a sensitivity of 0.188 ng/mL (Finetest, EH1729).

Techniques:

Journal: Brain and Behavior

Article Title: Relationship of Autophagic Dysfunction With the Quality of Life and Sleep, Depression and Disease Severity in Patients With Restless Legs Syndrome

doi: 10.1002/brb3.70172

Figure Lengend Snippet: The receiver operating characteristic curve analyze results and optimal cut‐off levels of ATG3 and ATG5 in patients with restless legs syndrome.

Article Snippet: The ATG5 measure has a sensitivity of 0.188 ng/mL (Finetest, EH1729).

Techniques: