Journal: Antioxidants (Basel, Switzerland)

Article Title: Rubicon, a Key Molecule for Oxidative Stress-Mediated DNA Damage, in Ovarian Granulosa Cells.

doi: 10.3390/antiox14040470

Figure Lengend Snippet: Figure 3. Effects of Trehalose treatment on autophagy-related proteins in HGrC1 cells. (A) Western blotting (WB) analysis evaluating LC3 expression levels in response to various autophagy activators in HGrC1 cells. The cells were treated for 24 h with Torin1 (1 nM), T-B1 (40 µM), Rapamycin (Rapa, 1 µM), Resveratrol (Resv, 100 µM), Curcumin (CCM, 10 µM), and Trehalose (Tre, 100 mM). (B) Cell viability assessed using the WST-1 assay in HGrC1 cells with various concentrations of Tre for 24 h. The Y-axis represented the mean fluorescence intensity, and the X-axis showed the Tre concentration (mM). (C) WB analysis of LC3 expression in HGrC1 cells treated with 100 mM maltose (Mal), 100 mM Tre, or 200/100 mM glucose (Glu) (mM) for 24 h. Actin was used as the control. The control cells were cultured in a medium only. (D) WB analysis of autophagy flux in HGrC1 cells treated with Trehalose (Tre) for 24 h. Cells were exposed to 100 mM Tre for 24 h, followed by chloroquine (CQ, 100 µM) treatment for 2 h before harvest. (E) Quantification of LC3-II expression based on the WB data shown in (D), demonstrating the effect of Tre and CQ on autophagy flux. (F) WB analysis of autophagy-related proteins in HGrC1 cells treated with 100 mM Tre for 24 h. WB were shown as follows: Rubicon, Beclin1, LC3, p62, UVRAG, Ulk1, TFEB, ATG3, ATG5, ATG14, and Actin. (G) WB analysis of Rubicon expression in HGrC1 cells treated with 100 mM Maltose (Mal), 100 mM Trehalose (Tre), or 200/100 mM Glucose (Glu) for 24 h. Actin was used as the loading control. Control cells were cultured in medium only. (H) Quantification of Rubicon expression based on the Western blot data shown in (G), comparing its levels across different treatment conditions. Results were obtained from at least three independent experiments. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Primary antibodies used in Western blotting were as follows: Actin (1:10,000, 3700, Cell-Signaling Technology (CST), Danvers, MA, USA), ATG3 (1:2000, TA503346, OriGene, Rockville, MD, USA), ATG5 (1:2000, M153-3MS, MBL Life science, Tokyo, Japan), ATG14 (1:2000, 5504, CST), Ataxia Telangiectasia Mutated (ATM, 1:5000, 2873, CST), p-ATM (Ser1981, 1:5000, 5883, CST), Beclin1 (1:2000, sc48381, Antioxidants 2025, 14, 470 4 of 19 Santa Cruz Biotechnology, Santa Cruz, TX, USA), BNIP3L (1:2000, 12986-1-AP, Proteintech, Rosemont, IL, USA), γH2A histone family member X (γH2AX Ser139, 1:2000, 9718, CST), Microtubule Associated Protein 1 Light Chain 3 beta (LC3, 1:2000, 76446, Proteintech), MFN1 (1:4000, 14739, CST), MFN2 (1:4000, 11925, CST), mTOR (1:4000, 2972, CST), pmTOR (1:4000, 2971, CST), p62 (1:5000, M162-3, MBL Life science), p-p62 (Ser403, 1:2000, GTX128171, GeneTex, Irvine, CA, USA), p95 (1:5000, 14956, CST), p-p95 (Ser343, 1:4000, 3001, CST), Rubicon (1:5000, 8465, CST), Tom20 (1:2000, 42406, CST), and ULK1 (1:2000, 8054, CST), UVRAG (1:2000, M160-3MS, MBL Life science).

Techniques: Western Blot, Expressing, WST-1 Assay, Fluorescence, Concentration Assay, Control, Cell Culture

Journal: EMBO reports

Article Title: eIF5A is required for autophagy by mediating ATG3 translation.

doi: 10.15252/embr.201846072

Figure Lengend Snippet: Figure 4. eIF5A regulates translation of ATG3.

Article Snippet: Primary antibodies used are as follows: LC3B (nanotools, 1:200) (LC3B CST, 1:1,000), GABARAP (Abgent 1:1,000), GATE-16 (MBL, 1:1,000), ATG3 (Sigma, 1:500), Vinculin (Sigma, 1:100,000), GAPDH (Santacruz, 1:20,000), eIF5A (Santacruz, 1:500), Lamin A1 (Santacruz, 1:1,000), histone H3 (Abcam 1:10,000), p62 (MBL, 1:5,000), RFP/ Cherry (Rockland, 1:6,000), p-mTOR (Ser 2448) (CST, 1:1,000), mTOR (Cell signaling, 1:1,000), RPL23A (Abcam, 1:50,000), RPS6 (CST, 1:1,000), RPL10A (Santacruz, 1:2,000), Hypusine (Merck Millipore 1:5,000).

Techniques:

Journal: EMBO reports

Article Title: eIF5A is required for autophagy by mediating ATG3 translation.

doi: 10.15252/embr.201846072

Figure Lengend Snippet: Figure 5. Regulation of autophagy by eIF5A is mediated via ATG3 translation.

Article Snippet: Primary antibodies used are as follows: LC3B (nanotools, 1:200) (LC3B CST, 1:1,000), GABARAP (Abgent 1:1,000), GATE-16 (MBL, 1:1,000), ATG3 (Sigma, 1:500), Vinculin (Sigma, 1:100,000), GAPDH (Santacruz, 1:20,000), eIF5A (Santacruz, 1:500), Lamin A1 (Santacruz, 1:1,000), histone H3 (Abcam 1:10,000), p62 (MBL, 1:5,000), RFP/ Cherry (Rockland, 1:6,000), p-mTOR (Ser 2448) (CST, 1:1,000), mTOR (Cell signaling, 1:1,000), RPL23A (Abcam, 1:50,000), RPS6 (CST, 1:1,000), RPL10A (Santacruz, 1:2,000), Hypusine (Merck Millipore 1:5,000).

Techniques:

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