Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Melatonin inhibits mTOR-dependent autophagy during liver ischemia/reperfusion.

doi: 10.1159/000356647

Figure Lengend Snippet: Fig. 4. Effect of MLT on autophagy during liver I/R. MLT was administered 15 min prior to ischemia and again directly before reperfusion. Western blot analysis was performed to measure protein levels of LC3- II/I (A), SQSTM1/p62 (), beclin-1 (C), Atg3 (E), and Atg7 (F). Autophagic vacuoles (arrows indicated) were observed from TEM images (4 mice per group) (D). Results are presented as the mean ± S.E.M. of 6 to 8 mice per group. *, ** Signiϐicantly different (p < 0.05, p < 0.01) from sham-operated animals. +, ++ Signiϐicantly different (p < 0.05, p < 0.01) from I/R animals, respectively.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, SA) eclin-1 (3738), Atg3 (3415), Atg7 (2631), p-mammalian target of rapamycin (mTOR) (Ser2448) (2971), p-4E- P1 (Ser65) (9451) and p-70S6K (Thr389) (9205).

Techniques: Western Blot

Journal: International journal of molecular medicine

Article Title: Tougu Xiaotong capsule promotes chondrocyte autophagy by regulating the Atg12/LC3 conjugation systems.

doi: 10.3892/ijmm.2014.1794

Figure Lengend Snippet: Figure 5. Tougu Xiaotong capsule (TXC) regulates the mRNA expression of autophagy-related (Atg) genes in cobalt chloride (CoCl2)-exposed chondro cytes. (A) mRNA expression of Atg genes in CoCl2-exposed chondrocytes treated with or without TXC measured by RT-PCR. The mRNA expression of (B) hypoxia-inducible factor-1α (HIF-1α), (C) beclin 1, (D) mammalian target of rapamycin (mTOR), (E) Atg3, (F) Atg5, (G) Atg7, (H) Atg10, (I) Atg12 and (J) microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I. β-actin was used as the internal control for the quantification analysis. Data are the means ± standard deviation (SD) and SD is shown as vertical bars, *P<0.05, **P<0.01, compared to untreated cells; ΔP<0.05, ΔΔP<0.01, compared to CoCl2- exposed-chondrocytes.

Article Snippet: The PVDF membrane was blocked with 5% non-fat milk and incubated with antibodies to HIF-1α, beclin 1, mTOR, Atg3, Atg7, Atg12, LC3 I/II and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Standard Deviation

Journal: International journal of molecular medicine

Article Title: Tougu Xiaotong capsule promotes chondrocyte autophagy by regulating the Atg12/LC3 conjugation systems.

doi: 10.3892/ijmm.2014.1794

Figure Lengend Snippet: Figure 6. Tougu Xiaotong capsule (TXC) regulates the protein expression of autophagy-related (Atg) genes in cobalt chloride (CoCl2)-exposed chondrocytes. (A) Protein expression of Atg genes in CoCl2-exposed chondrocytes measured by western blot analysis. Protein levels of (B) hypoxia-inducible factor-1α (HIF- 1α), (C) beclin 1, (D) mammalian target of rapamycin (mTOR), (E) Atg3, (F) Atg7, (G) Atg12, and (H) microtubule-associated protein 1 light chain 3 (LC3) II/ LC3 I. β-actin was used as the internal control for the quantification analysis. Data are the means ± standard deviation (SD) and SD is shown as vertical bars, *P<0.05, **P<0.01, compared to untreated cells; ΔP<0.05, ΔΔP<0.01, compared to CoCl2-exposed-chondrocytes.

Article Snippet: The PVDF membrane was blocked with 5% non-fat milk and incubated with antibodies to HIF-1α, beclin 1, mTOR, Atg3, Atg7, Atg12, LC3 I/II and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Nature Communications

Article Title: Oxidation of Atg3 and Atg7 mediates inhibition of autophagy

doi: 10.1038/s41467-017-02352-z

Figure Lengend Snippet: Under pro-oxidizing conditions a disulfide forms between the catalytic thiols of Atg3 and Atg7. a siRNA knock-down of Atg7 attenuates intermolecular disulfide oxidation of Atg3, and loss of Atg3 prevents Atg7 disulfide dimerization. b Immunoprecipitation of Atg7 leads co-purification of disulfide-bound mCherry-Atg3. c Overexpressed wild-type mCherryAtg3 and wild-type Atg7 form a disulfide-bound heterodimer in amino-acid-starved cells treated with H 2 O 2 . However, mutation of the catalytic thiol to an alanine on either Atg3 or Atg7 prevents intermolecular disulfide formation

Article Snippet: In some experiments, SMC were transfected for 48 h with a combination of 250 ng of wild-type mCherryAtg3 (Addgene, 54993), C264A mCherryAtg3, wild-type Atg7 (Sino Biological, HG15684) or C572A Atg7 using lipofectamine 3000.

Techniques: Knockdown, Immunoprecipitation, Copurification, Mutagenesis