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

Journal: The Journal of Biological Chemistry

Article Title: Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins

doi: 10.1074/jbc.AC119.009977

Figure Lengend Snippet: ATG3 protein is a target of LC3/GABARAP conjugation and ATG4-mediated deconjugation. A, enrichment of transiently expressed 3xFLAG–LC3B G120 from HeLa control and ATG4B KO cells by IP with anti-FLAG antibody prior to Western blotting with anti-FLAG antibody ( left ) and anti-ATG3 antibodies ( middle, right panel ). Cells were transfected with GFP as a negative control. B, 3xFLAG–LC3B G120 expressed in HeLa ATG4B KO cells was enriched by IP with anti-FLAG antibody prior to treatment with 0.02 mg/ml recombinant GST–ATG4B WT or C74S at 37 °C for 1 h and Western blotting using anti-FLAG and anti-ATG3 N-ter antibody. C, HeLa control and ATG4B KO cells were transfected with 3xFLAG–LC3B G120, 3xFLAG–LC3B Q116P G120, or GFP as a negative control prior to harvesting. Western blotting was performed to detect 3xFLAG–LC3B and ATG3 using the same lysates diluted with either nonreducing (β-mercaptoethanol excluded) or reducing (standard recipe) sample buffer. Thioester-linked species are detected under nonreducing conditions only. D, Western blotting of HeLa ATG4B KO GFP–LC3B G120 cells transiently transfected with 3xFLAG–ATG3 WT and point mutant constructs. Samples were lysed in buffer lacking NEM and processed under reducing conditions ( upper panel ). The same constructs were used to rescue HeLa ATG3 KO cells treated for 3 h with 250 n m Torin1 and 10 n m baf A1 and assessed by Western blotting under nonreducing conditions ( lower panel ). E, schematic of human ATG4 protease cellular function in protein deconjugation ( deLC3ylation ), which counteracts covalent attachment of LC3/GABARAP (LC3ylatio n ) to ATG3 protein at residue Lys-243.

Article Snippet: To generate an ATG3 entry clone encoding WT ATG3 with a stop codon, the ATG3 CDS was PCR-amplified from pDONR223_ATG3_WT_V5 (a gift from Jesse Boehm, Matthew Meyerson, and David Root, Addgene plasmid no. 82952 ( )) using the primers ATG3 forward (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCAGAATGTGATTAATACTGTG-3′) and ATG3 reverse with stop codon (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTATTACATTGTGAAGTGTCTTGTGTAG-3′).

Techniques: Conjugation Assay, Control, Western Blot, Transfection, Negative Control, Recombinant, Mutagenesis, Construct, Cell Function Assay, Residue

Journal: Autophagy

Article Title: ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

doi: 10.1080/15548627.2016.1212786

Figure Lengend Snippet: Lack of ATG3-dependent mitochondrial autophagy leads to defective reprogramming. (A) Western blot analysis of whole cell extracts from Atg3+/+ and atg3−/− MEFs; ACTB served as a loading control. (B) Mitophagy in Atg3+/+ and atg3−/− MEFs during iPSC induction. Blue: Hoechst 33342; Red: MitoTracker Red; Green: GFP-MAP1LC3B. Colocalizations of MAP1LC3B and mitochondria were counted in 50 cells. Data shown as mean ± standard deviation (SD), n = 3; **, P < 0.01; Student t test. (C) Representative transmission electronic microscopy images of autophagic mitochondria in reprogramming Atg3+/+ and atg3−/− MEFs. Bar: 2 μm. (D) Mitochondrial decreases were impaired by Atg3 deletion during reprogramming. Data normalized toatg3−/− cells and shown as mean ± standard deviation (SD), n = 3; *, P < 0.05; ***, P < 0.001; Student t test. (E) Defective autophagy in atg3−/− MEFs was rescued by reacquisition of wild-type Atg3 expression. The indicated cells were starved for 2 h and then harvested for western blot using anti-ATG3, anti-MAP1LC3B, anti-SQSTM1 and anti-ACTB antibodies. (F) Elevated Mito-mass in atg3−/− reprogramming MEFs was recovered by wild-type Atg3 expression. Data normalized to Atg3+/+ cells and shown as mean ± SD, n = 3; **, P < 0.01; Student t test; NS, not significant. (G) Reacquisition of wild-type but not V8D mutant Atg3 expression in atg3−/− cells compensated for reprogramming efficiency. Data shown as mean ± SD, n = 3; **, P < 0.01; ***, P < 0.001; Student t test. (H) Representative transmission electronic microscopy images of mitochondria in Atg3+/+ and atg3−/− iPSCs, Bar: 1 μm. (I) Mitochondrial membrane potential was significantly reduced in atg3−/− iPSCs in contrast to Atg3+/+ iPSCs. Data normalized to Atg3+/+ cells and shown as mean ± standard deviation (SD), n = 3; **, P < 0.01; Student t test. (J) atg3−/− iPSCs have a significantly lower oxygen consumption rate than Atg3+/+ iPCSs. Data normalized to Atg3+/+ cells and shown as mean ± SD, n = 3; **, P < 0.01; Student t test. (K) atg3−/− iPSCs have a significant lower ATP level than Atg3+/+ iPCSs. Data normalized to Atg3+/+ cells and shown as mean ± SD, n = 3; *, P < 0.05; Student's t test.

Article Snippet: Animals, reagents, antibodies, and plasmids B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb (RBRC03743), 35 MAP1LC3B-GFP (RBRC00806), 36 and Atg3 +/− (RBRC02761) 16 mice were purchased from Riken BioResource Center.

Techniques: Western Blot, Control, Standard Deviation, Transmission Assay, Microscopy, Expressing, Mutagenesis, Membrane

Journal: Autophagy

Article Title: ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

doi: 10.1080/15548627.2016.1212786

Figure Lengend Snippet: ATG3 regulates ESC mitochondrial homeostasis and self-renewal. (A) Western blot analysis of whole cell extracts from Atg3+/+ and atg3−/− ESCs; ACTB served as a loading control. (B) Lack of FCCP-induced mitophagy in atg3−/− ESCs. Green: MAP1LC3B; Red: MitoTracker Red; Blue: Hoechst 33342. Colocalizations of LC3 and mitochondria were counted in 50 cells. Data shown as mean ± SD, n = 3; **, P < 0.01; Student t test. (C) Mitochondria accumulated in atg3−/− ESCs. Data presented as the mean of 3 independent experiments normalized to Atg3+/+ ESCs and shown as mean ± standard deviation (SD), n = 3; ***, P < 0.001; Student t test. (D) Transmission electronic microscopy images of mitochondria in Atg3+/+ and atg3−/− ESCs. (E) Increased mtDNA copy number in atg3−/− ESCs was rescued by gain of wild-type but not V8D mutant Atg3 expression. Data were normalized to Atg3+/+ ESCs and shown as mean ± SD, n = 3; *, P < 0.05; **, P < 0.01; ***P < 0.001; Student t test. (F) The enhanced ROS generation in atg3−/− ESCs was rescued by reacquisition expression of WT but not V8D mutant Atg3. Data normalized to Atg3+/+ ESCs and shown as mean ± SD, n = 3; *, P < 0.05; **, P < 0.01; Student t test. (G) Defective ATP generation in atg3−/− ESCs was partially rescued by gain of wild-type but not V8D mutant Atg3. Data normalized to Atg3+/+ ESCs and shown as mean ± SD, n = 3; *, P < 0.05; ***, P < 0.001; Student t test. (H) Aberrant self-renewal of atg3−/− ESCs was partially rescued by reacquisition of wild-type but not V8D mutant Atg3 expression. Data shown as mean ± SD, n = 3; **, P < 0.01; ***, P < 0.001; Student's t test.

Article Snippet: Animals, reagents, antibodies, and plasmids B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb (RBRC03743), 35 MAP1LC3B-GFP (RBRC00806), 36 and Atg3 +/− (RBRC02761) 16 mice were purchased from Riken BioResource Center.

Techniques: Western Blot, Control, Standard Deviation, Transmission Assay, Microscopy, Mutagenesis, Expressing

Journal: Autophagy

Article Title: ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

doi: 10.1080/15548627.2016.1212786

Figure Lengend Snippet: Loss of ATG3-mediated mitochondrial autophagy compromises ESC pluripotency and differentiation. (A) Mitochondrial mass (Mito-mass) increased during EB differentiation. Data presented as the mean ± error from 3 independent experiments. (B) Mitochondrial mass increases during EB differentiation were impaired by Atg3 deletion. Mean values were normalized to Atg3+/+ EB, n = 3; *, P < 0.05; Student t test. (C) Atg3+/+ and atg3−/− ESC-formed EBs were analyzed by JC-1 staining at the indicated differentiation times. Mean values were normalized to Atg3+/+ EB, n = 3; *, P < 0.05; Student t test. (D) Transcription of pluripotency genes in ESCs was impaired by Atg3 deletion. Data are representative of 3 independent experiments. (E) Lack of ATG3-dependent mitophagy impaired ESC lineage specification. Data shown are representative of 3 independent experiments. (F) Atg3 deletion compromised ESC teratoma differentiation. Data from 3 independent experiment and shown as mean± SD.

Article Snippet: Animals, reagents, antibodies, and plasmids B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb (RBRC03743), 35 MAP1LC3B-GFP (RBRC00806), 36 and Atg3 +/− (RBRC02761) 16 mice were purchased from Riken BioResource Center.

Techniques: Staining

Journal: Cancer Management and Research

Article Title: ATG3, a Target of miR-431-5p, Promotes Proliferation and Invasion of Colon Cancer via Promoting Autophagy

doi: 10.2147/CMAR.S226828

Figure Lengend Snippet: ATG3 is upregulated in colon cancer tissues and cells. Notes: Upregulation of ATG3 in colon cancer is supported by online data from TCGA ( A ) and GEO database ( B and C ). Upregulation of ATG3 in colon cancer is confirmed by qPCR ( D ) and IHC ( E ) in our collected tissues. COAD: colon adenocarcinoma, “Colon” and “Para-cancer” means normal tissues in this part. *Stands for P < 0.05; ***Stands for P < 0.001.

Article Snippet: Specific siRNAs targeting ATG3 at 3ʹ-untranslated region and miR-431-5p mimics were synthesized by RiboBio Inc (Guangzhou, China).

Techniques:

Journal: Cancer Management and Research

Article Title: ATG3, a Target of miR-431-5p, Promotes Proliferation and Invasion of Colon Cancer via Promoting Autophagy

doi: 10.2147/CMAR.S226828

Figure Lengend Snippet: ATG3 knockdown inhibits proliferation and invasion of colon cancer cells. Notes: qPCR ( A ) and Western blot ( B ) show that ATG3 is upregulated in colon cancer cells compared with colon epithelial cell NCM460. ( C ) Western blot indicates that ATG3 is successfully knocked down in SW620 and HCT116 cells. ATG3 knockdown significantly suppresses proliferation and invasion of colon cancer cells demonstrated by plate clone formation ( D ) and Transwell assays ( E ). siNC stands for negative control small RNA; Ns stands for no significant difference; **Stands for P < 0.01; ***Stands for P < 0.001.

Article Snippet: Specific siRNAs targeting ATG3 at 3ʹ-untranslated region and miR-431-5p mimics were synthesized by RiboBio Inc (Guangzhou, China).

Techniques: Knockdown, Western Blot, Negative Control

Journal: Cancer Management and Research

Article Title: ATG3, a Target of miR-431-5p, Promotes Proliferation and Invasion of Colon Cancer via Promoting Autophagy

doi: 10.2147/CMAR.S226828

Figure Lengend Snippet: miR-431-5p is downregulated, inversely correlates with ATG3, and directly targets ATG3 in colon cancer. Notes: ( A and B ) miR-431-5p is downregulated in colon cancer tissues and cells indicated by qPCR assay. ( C ) miR-431-5p expression is inversely associated with that of ATG3 in colon cancer indicating by Pearson correlations analysis ( r = −0.5773, P <0.01). ( D ) Sketch of wild and mutant luciferase reports plasmids and dual luciferase reporter system assay shows that miR-432-5p can directly target ATG3. (E) Western blot shows that ATG3 can be inhibited by miR-431-5p mimics, which can be reversed by ectopic expression of ATG3. **Stands for P < 0.01; ***Stands for P < 0.001.

Article Snippet: Specific siRNAs targeting ATG3 at 3ʹ-untranslated region and miR-431-5p mimics were synthesized by RiboBio Inc (Guangzhou, China).

Techniques: Expressing, Mutagenesis, Luciferase, Western Blot

Journal: Cancer Management and Research

Article Title: ATG3, a Target of miR-431-5p, Promotes Proliferation and Invasion of Colon Cancer via Promoting Autophagy

doi: 10.2147/CMAR.S226828

Figure Lengend Snippet: miR-431-5p could inhibit proliferation and invasion of colon cancer cells by targeting ATG3. Notes: ( A ) As the results of plate clone formation assay indicated, miR-431-5p mimic significantly inhibits proliferation of SW620 and HCT116 which could be subsequently rescued by restoration of ATG3. ( B ) miR-431-5p mimic significantly inhibits invasion of SW620 and HCT116 which could be subsequently rescued by ectopic expression of ATG3, demonstrating by Transwell assay. **Stands for P < 0.01; ***Stands for P < 0.001.

Article Snippet: Specific siRNAs targeting ATG3 at 3ʹ-untranslated region and miR-431-5p mimics were synthesized by RiboBio Inc (Guangzhou, China).

Techniques: Tube Formation Assay, Expressing, Transwell Assay

Journal: Cancer Management and Research

Article Title: ATG3, a Target of miR-431-5p, Promotes Proliferation and Invasion of Colon Cancer via Promoting Autophagy

doi: 10.2147/CMAR.S226828

Figure Lengend Snippet: ATG3 promotes proliferation and invasion of colon cancer cells in an autophagy-dependent manner. Notes: ( A ) ATG3 knockdown inhibits autophagy indicated by less LC3B accumulation with or without CQ (50μM) addition. ( B ) Ectopic expression of ATG3 restores autophagy flux of colon cancer cells demonstrated by LC3B accumulation. ( C and D) CQ (50μM) treatment antagonizes the promotive effects of ATG3 on proliferation and invasion of colon cancer cells indicated by plate clone formation and Transwell assays. **Stands for P < 0.01; ***Stands for P < 0.001.

Article Snippet: Specific siRNAs targeting ATG3 at 3ʹ-untranslated region and miR-431-5p mimics were synthesized by RiboBio Inc (Guangzhou, China).

Techniques: Knockdown, Expressing

Journal: Nature Communications

Article Title: An N-terminal conserved region in human Atg3 couples membrane curvature sensitivity to conjugase activity during autophagy

doi: 10.1038/s41467-020-20607-0

Figure Lengend Snippet: a Sequence analysis of Atg3 from multiple species, as well as the consensus sequence. All sequences begin at the first residue of the protein; residues numbered at the top refer to the human sequence. Non-polar and aromatic residues are in yellow, polar residues in green, positively charged and histidine residues in blue, negatively charged residues in red, alanine and glycine residues in black, and proline residue in purple. b Diagrams of hAtg3 NT constructs prepared in this study. Sequences corresponding to the hAtg3 sequence are teal and mutations and insertions are gold.

Article Snippet: Atg3 human (MQNVINTVKGKALEVAEYLTPVLKESK) and yeast (MIRSTLSSWREYLTPITHKST) NTs and hAtg3 NT mutants (A12W, A12W_P21A, A12W_L23T, A12W_V8D_V15K) were synthesized by GenScript (Piscataway, New Jersey) at >95% purity as determined by high-performance liquid chromatography (HPLC) and mass spectroscopy (MS).

Techniques: Sequencing, Residue, Construct

Journal: Nature Communications

Article Title: An N-terminal conserved region in human Atg3 couples membrane curvature sensitivity to conjugase activity during autophagy

doi: 10.1038/s41467-020-20607-0

Figure Lengend Snippet: Atg3 knockout (Atg3 −/− ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry, mCherry-hAtg3, mCherry-hAtg3 P21A , or mCherry-hAtg3 L23T were cultured in complete media (CM) and starvation media (SM) with or without bafilomycin A1 (BafA1) for 3 h and subjected to immunoblotting with the indicated antibodies. a Representative immunoblot ( n = 6 blots). b Quantitative analysis of the relative LC3B–II level ( n = 6 blots). c Relative basal and starvation-induced autophagic flux ( n = 6 blots). Statistical analysis was performed using one-way ANOVA test followed by Dunnett’s multiple comparisons test. Data are presented as mean ± SD. P values: **** P < 0.0001 ( b ), *** P = 0.0001 and 0.0003 (left to right in b ), ** P = 0.0037 and 0.0011 (left to right in b ) and 0.0014 and 0.0021 (left to right in c ), and * P = 0.0097 ( c ).

Article Snippet: Atg3 human (MQNVINTVKGKALEVAEYLTPVLKESK) and yeast (MIRSTLSSWREYLTPITHKST) NTs and hAtg3 NT mutants (A12W, A12W_P21A, A12W_L23T, A12W_V8D_V15K) were synthesized by GenScript (Piscataway, New Jersey) at >95% purity as determined by high-performance liquid chromatography (HPLC) and mass spectroscopy (MS).

Techniques: Knock-Out, Stable Transfection, Expressing, Cell Culture, Western Blot