Journal: Molecular cell

Article Title: VPS34 Acetylation Controls Its Lipid Kinase Activity and the Initiation of Canonical and Non-canonical Autophagy.

doi: 10.1016/j.molcel.2017.07.024

Figure Lengend Snippet: Figure 1. VPS34 Is Acetylated by p300 at K29, K771, and K781 (A) Acetylation of VPS34 in HEK293 cells treated with the deacetylase inhibitors TSA and NAM. VPS34 was immunoprecipitated from cell lysates with an anti-VPS34 antibody, then analyzed by immunoblot using an anti-acetyl-lysine antibody (Ace-lys). (B and C) VPS34 acetylation in HEK293 cells overexpressing the indicated individual histone acetyltransferases (B) or treated with the p300 activator CTB or the p300 inhibitor C646 (C). H3, Histone3; Ace-H3, acetyl-histone3 (Lys56). (D) VPS34 acetylation in HEK293 cells transfected with p300-HA or the acetyltransferase-dead p300-WY-HA after 48 hr incubation with p300 siRNA. (E) Association of p300 with VPS34. VPS34 was immunoprecipitated from HEK293 cells treated with or without the cross-linker DPDPB, and the precipitates were analyzed using anti-p300. (F) In vitro acetylation assays using purified GST-VPS34 and HA-tagged p300 or p300-WY immunoprecipitated from treated HEK293T cells. (G) In vitro acetylation assays using purified GST-VPS34 and p300 HAT domain. The arrow indicates the band of p300-HAT domain. Asterisk indicates non- specific band. (H and I) Acetylation of Flag-tagged VPS34 or VPS34 mutants expressed in cells. 5KR, all five Lys residues were replaced by Arg; 3KR, Lys 29, Lys 771, and Lys 781 were replaced by Arg. H.E., high exposure.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-HA affinity beads Biotool Cat#B23302 Rabbit ployclonal anti-Rubicon Abcam Cat#ab92388 Rabbit monoclonal anti-ATG7 Abcam Cat#ab133528 Rabbit ployclonal anti-Rab5 Abcam Cat#ab18211 Mouse monoclonal anti-Rab7 Abcam Cat#ab50533 Rabbit monoclonal anti-EGFR Abcam Cat#ab52894 Mouse monoclonal anti-ATG5 MBL Cat#M153-3 Bacterial and Virus Strains E. coli BL21 Transgen Biotech Cat#CD601 Trans 5a Transgen Biotech Cat#CD201 Chemicals, Peptides, and Recombinant Proteins Trichostatin A Sigma-Aldrich Cat#T1952 Nicotinamide Sigma-Aldrich Cat#72340 CTB Sigma-Aldrich Cat#C6499 C646 Selleck Cat#S7152 g-32P-ATP PerkinElmer Cat#NEG502A 3-methyladenine (3-MA) Sigma-Aldrich Cat#M9281 Torin1 Selleck Cat#S2827 Tissue-Tek OCT compound Sakura Cat#4583 Cocktail Roche Cat#04693132001 DPDPB (1,4-bis(3-(2-pyridyldithio) propionamido butane) Sigma-Aldrich Cat#16646 Acetyl-coenzyme A Sigma-Aldrich Cat#A2056 Glucose-free DMEM Life Technologies Cat#11966025 Lipofectamine 2000 Invitrogen Cat#11668019 UK5099 (2-Cyano-3-(1-phenyl-1H-indol3-yl) 2-propenoic acid) Sigma Cat#PZ0160 Dichloroacetate (DCA) Sigma Cat#347795 I-CBP112 Sigma Cat#SML1134 SGC-CBP30 Selleck Cat#S7256 p300-HAT Upstate Cat#14-418 Critical Commercial Assays BCA protein assay kit Thermo Cat#23227 PicoProbe Acetyl CoA Assay Kit (Fluorometric) Abcam Cat#ab87546 Class III PI3-Kinase ELISA Assay Kit Echelon Cat#K-3000 Deposited Data Original images were deposited to Mendeley data This paper http://dx.doi.org/10.17632/ x3tsv9hjx3.1 Experimental Models: Cell Lines

Techniques: Histone Deacetylase Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro

Journal: Molecular cell

Article Title: VPS34 Acetylation Controls Its Lipid Kinase Activity and the Initiation of Canonical and Non-canonical Autophagy.

doi: 10.1016/j.molcel.2017.07.024

Figure Lengend Snippet: Figure 2. Acetylation by p300 Inactivates VPS34 (A) Distribution of GFP-FYVE in HEK293 cells treated with the p300 activator CTB or the p300 inhibitor I-CBP112, SGC-CBP30, or C646, with or without the VPS34 inhibitor 3-MA. (B) GFP-FYVE distribution in p300-HA-overexpressing or p300-KO HEK293 cells. (C) Quantification of GFP-FYVE puncta in HEK293 cells overexpressing different acetyltransferases. (D and E) In vitro lipid kinase assays of VPS34 immunoprecipitated from C646- and CTB-treated HEK293 cells (D), and from p300-KO HEK293 cells with or without p300-HA or p300-WY-HA transfection (E). (F) Quantification of GFP-FYVE puncta in HEK293 cells co-transfected with plasmids expressing WT VPS34 and different KR and KQ mutants. (G and H) In vitro lipid kinase assays of the Flag-tagged VPS34 mutants immunopurified from transfected HEK293T cells. H.E., high exposure. Relative ratios of the 32P-PI3P signal divided by the VPS34 protein signal are shown in (D), (E), (G), and (H). Scale bar, 10 mm. The statistical results are shown as mean ± SEM, n = 30. ***p < 0.001. 3KR, Lys 29, Lys 771, and Lys 781 were replaced by Arg; 3KQ, Lys 29, Lys 771, and Lys 781 were replaced by Gln.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-HA affinity beads Biotool Cat#B23302 Rabbit ployclonal anti-Rubicon Abcam Cat#ab92388 Rabbit monoclonal anti-ATG7 Abcam Cat#ab133528 Rabbit ployclonal anti-Rab5 Abcam Cat#ab18211 Mouse monoclonal anti-Rab7 Abcam Cat#ab50533 Rabbit monoclonal anti-EGFR Abcam Cat#ab52894 Mouse monoclonal anti-ATG5 MBL Cat#M153-3 Bacterial and Virus Strains E. coli BL21 Transgen Biotech Cat#CD601 Trans 5a Transgen Biotech Cat#CD201 Chemicals, Peptides, and Recombinant Proteins Trichostatin A Sigma-Aldrich Cat#T1952 Nicotinamide Sigma-Aldrich Cat#72340 CTB Sigma-Aldrich Cat#C6499 C646 Selleck Cat#S7152 g-32P-ATP PerkinElmer Cat#NEG502A 3-methyladenine (3-MA) Sigma-Aldrich Cat#M9281 Torin1 Selleck Cat#S2827 Tissue-Tek OCT compound Sakura Cat#4583 Cocktail Roche Cat#04693132001 DPDPB (1,4-bis(3-(2-pyridyldithio) propionamido butane) Sigma-Aldrich Cat#16646 Acetyl-coenzyme A Sigma-Aldrich Cat#A2056 Glucose-free DMEM Life Technologies Cat#11966025 Lipofectamine 2000 Invitrogen Cat#11668019 UK5099 (2-Cyano-3-(1-phenyl-1H-indol3-yl) 2-propenoic acid) Sigma Cat#PZ0160 Dichloroacetate (DCA) Sigma Cat#347795 I-CBP112 Sigma Cat#SML1134 SGC-CBP30 Selleck Cat#S7256 p300-HAT Upstate Cat#14-418 Critical Commercial Assays BCA protein assay kit Thermo Cat#23227 PicoProbe Acetyl CoA Assay Kit (Fluorometric) Abcam Cat#ab87546 Class III PI3-Kinase ELISA Assay Kit Echelon Cat#K-3000 Deposited Data Original images were deposited to Mendeley data This paper http://dx.doi.org/10.17632/ x3tsv9hjx3.1 Experimental Models: Cell Lines

Techniques: In Vitro, Immunoprecipitation, Transfection, Expressing

Journal: Molecular cell

Article Title: VPS34 Acetylation Controls Its Lipid Kinase Activity and the Initiation of Canonical and Non-canonical Autophagy.

doi: 10.1016/j.molcel.2017.07.024

Figure Lengend Snippet: Figure 6. p300-mediated VPS34 Acetylation in Non-canonical Autophagy (A and B) Quantification of GFP-DFCP1 puncta (A) and LC3 puncta (B) in gene-deleted MEFs and Beclin 1-deleted HEK293 cells with or without C646 treatment or p300 RNAi. Data are presented as mean ± SEM, n = 30. (C) Immunoblot assay of intracellular p62 and LC3 in the gene-deleted MEFs. The cells were treated with p300 siRNA with or without CQ (10 mM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-HA affinity beads Biotool Cat#B23302 Rabbit ployclonal anti-Rubicon Abcam Cat#ab92388 Rabbit monoclonal anti-ATG7 Abcam Cat#ab133528 Rabbit ployclonal anti-Rab5 Abcam Cat#ab18211 Mouse monoclonal anti-Rab7 Abcam Cat#ab50533 Rabbit monoclonal anti-EGFR Abcam Cat#ab52894 Mouse monoclonal anti-ATG5 MBL Cat#M153-3 Bacterial and Virus Strains E. coli BL21 Transgen Biotech Cat#CD601 Trans 5a Transgen Biotech Cat#CD201 Chemicals, Peptides, and Recombinant Proteins Trichostatin A Sigma-Aldrich Cat#T1952 Nicotinamide Sigma-Aldrich Cat#72340 CTB Sigma-Aldrich Cat#C6499 C646 Selleck Cat#S7152 g-32P-ATP PerkinElmer Cat#NEG502A 3-methyladenine (3-MA) Sigma-Aldrich Cat#M9281 Torin1 Selleck Cat#S2827 Tissue-Tek OCT compound Sakura Cat#4583 Cocktail Roche Cat#04693132001 DPDPB (1,4-bis(3-(2-pyridyldithio) propionamido butane) Sigma-Aldrich Cat#16646 Acetyl-coenzyme A Sigma-Aldrich Cat#A2056 Glucose-free DMEM Life Technologies Cat#11966025 Lipofectamine 2000 Invitrogen Cat#11668019 UK5099 (2-Cyano-3-(1-phenyl-1H-indol3-yl) 2-propenoic acid) Sigma Cat#PZ0160 Dichloroacetate (DCA) Sigma Cat#347795 I-CBP112 Sigma Cat#SML1134 SGC-CBP30 Selleck Cat#S7256 p300-HAT Upstate Cat#14-418 Critical Commercial Assays BCA protein assay kit Thermo Cat#23227 PicoProbe Acetyl CoA Assay Kit (Fluorometric) Abcam Cat#ab87546 Class III PI3-Kinase ELISA Assay Kit Echelon Cat#K-3000 Deposited Data Original images were deposited to Mendeley data This paper http://dx.doi.org/10.17632/ x3tsv9hjx3.1 Experimental Models: Cell Lines

Techniques: Western Blot