Journal: EMBO Reports

Article Title: UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation

doi: 10.1038/embor.2011.79

Figure Lengend Snippet: UVRAG inhibits BAX-induced apoptosis. (A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P<0.05). (B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 and -9 activation in HL60 cells were analysed (n=3, *P<0.05). (C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P<0.05). (D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment-induced apoptosis (n=3, *P<0.05). AU, arbitrary units; BAX, BCL2-associated X protein; shRNA, short-hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.

Article Snippet: The antibodies to UVRAG, cytochrome c and FAS were obtained from Santa Cruz Technology (Santa Cruz, CA, USA).

Techniques: Control, shRNA, Transfection, Plasmid Preparation, Membrane, Activation Assay, Knockdown, Irradiation

Journal: EMBO Reports

Article Title: UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation

doi: 10.1038/embor.2011.79

Figure Lengend Snippet: UVRAG interacts with Bax. (A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co-immunoprecipitation (RIPA or CHAPS buffer) or western blotting. (C) Proteins in the pellet after GST pull-down assays in HCT116 cell lysate were analysed by SDS–PAGE and immunodetected with UVRAG or GST antibodies. (D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag-UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot. (E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2-associated X protein; CCD, coiled-coil domain; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate; GST, glutathione-S-transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.

Article Snippet: The antibodies to UVRAG, cytochrome c and FAS were obtained from Santa Cruz Technology (Santa Cruz, CA, USA).

Techniques: Irradiation, Expressing, Immunoprecipitation, Western Blot, SDS Page, Binding Assay, Transfection, Radio Immunoprecipitation, Polyacrylamide Gel Electrophoresis

Journal: EMBO Reports

Article Title: UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation

doi: 10.1038/embor.2011.79

Figure Lengend Snippet: UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A–C) and activation of BAX (D–F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P<0.05. AU, arbitrary units; BAX, BCL2-associated X protein; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short-hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.

Article Snippet: The antibodies to UVRAG, cytochrome c and FAS were obtained from Santa Cruz Technology (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Transfection, shRNA, Irradiation, Western Blot, Activation Assay, Immunoprecipitation

Journal: PLoS Genetics

Article Title: Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

doi: 10.1371/journal.pgen.1004626

Figure Lengend Snippet: A . Hela cells treated with indicated siRNA (siCon is siControl; siBec is siBeclin 1) were treated with DMSO or Bafilomycin A1 (BafA). Representative composite fluorescence plots and normalized quantification of mean fluorescence intensity of 10,000 cells were determined from three separate experiments. Mean pHrodo dextran (PE-A) fluorescence; bars represent mean +/− s.e.m. (n = 3). DMSO pHrodo dextran siCon vs. siBec one sample t-test (two-tailed) p = 0.0283; BafA pHrodo dextran siCon vs. siBec one-tailed t-test p = 0.0246. B . Mean LysoSensor (Am Cyan-A) fluorescence intensity; bars represent mean +/− s.e.m. (n = 3). LysoSensor siCon vs. siBec one sample t-test (two-tailed) p = 0.0029. C . Anti-UVRAG, -Beclin 1, -Actin and –LC3 blots of HeLa cells lysed after indicated siRNA knock-down. Asterix indicates unspecific band. D . Receptor-mediated endocytosis as measured by EGFR internalization is inhibited in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. Anti-EGFR, - UVRAG, -beclin 1 and –actin blots of control or Becn1 deficient MEFs lysed after indicated treatment with EGF. Asterix indicates unspecific band. Quantification of normalized EGFR/actin from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3); p = 0.0395, 0.0208 (one-tailed t-test).

Article Snippet: Immunoprecipitation was performed with 500 μg protein from post-nuclear supernatant, using antibodies against VPS34 (Echelon), UVRAG (Bethyl), or ATG14 (MBL) overnight.

Techniques: Fluorescence, Two Tailed Test, One-tailed Test, Knockdown, Control

Journal: PLoS Genetics

Article Title: Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

doi: 10.1371/journal.pgen.1004626

Figure Lengend Snippet: A . Table showing results from PI(3)P ELISA. Average 450 nm absorbance, p-value of comparison to Becn1 deficient MEFs. PI(3)P concentration is determined by comparison to a standard curve. Quantification of 450 nm absorbance of control, Becn1 deficient, and Becn1 revertant MEFs (6,5,6 replicates); statistics using a one-tailed t-test p = 0.0032, 0.0336. B . Anti-beclin 1, and -actin blots of MEF cell lysates. Con is control MEFs, def is Becn1 deficient MEFs, and rev is Becn1 revertant MEFs plus beclin 1. C . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -beclin 1, and -actin blots after VPS34 IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. D . Quantification of normalized 32 P-labelled PI(3)P and 32 P-PI(3)P/VPS34 (from IP) from 3 separate experiments and VPS34/actin (from input lanes) from 9 separate experiments (including ). Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0003, p = 0.0226, and p = 0.0002 top to bottom using a one sample t-test (two-tailed). UVRAG and Atg14-associated PI(3)P production is decreased in Becn1 deficient MEFs. E . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -UVRAG, -beclin 1, and -actin blots after UVRAG IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p<0.0001 using a one sample t-test (two-tailed). F . Autoradiograph of 32 P-PI(3)P and anti-VPS34, -Atg14L, -beclin 1, and -actin blots after Atg14L IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0065 using a one sample t-test (two-tailed).

Article Snippet: Immunoprecipitation was performed with 500 μg protein from post-nuclear supernatant, using antibodies against VPS34 (Echelon), UVRAG (Bethyl), or ATG14 (MBL) overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Concentration Assay, Control, One-tailed Test, Autoradiography, Two Tailed Test

Journal: PLoS Genetics

Article Title: Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

doi: 10.1371/journal.pgen.1004626

Figure Lengend Snippet: A–E . Dispersion of GFP-p40 phox in Becn1 deficient MEFs is rescued with reintroduction of beclin 1 as well as overexpression of UVRAG but not Atg14L or RUBICON. Images of Becn1 deficient MEFs cotransfected with GFP-p40 phox and AsRed ( A ), beclin 1-AsRed ( B ), Atg14L-AsRed ( C ), RUBICON-AsRed ( D ) and UVRAG-Flag and stained with anti-Flag antibody ( E ). Scale bars = 10 µm. Quantification as in E. AsRed (5,26) vs. beclin 1-AsRed (28,3) significantly different (p<0.0001); AsRed (5,26) vs. Atg14L-AsRed (3,29) not significantly different (p = 0.4741); AsRed (5,26) vs. RUBICON-AsRed (5,27) not significantly different (p = 1.0000); AsRed (5,26) vs. UVRAG-Flag (26,4) significantly different (p<0.0001) all using Fisher's exact test (two-sided). Rescue of the aberrant GFP-p40 phox distribution in Becn1 deficient MEFs by beclin 1 overexpression requires the CC domain of beclin 1. A beclin 1 mutant lacking the CC domain does not rescue the GFP-p40 phox phenotype of Becn1 deficient MEFs. Immunofluorescent images of Becn1 deficient MEFs transfected with beclin 1-myc wt ( F ) or beclin 1-myc minus its UVRAG-binding CC (beclin 1ΔCCD-myc) ( G ) then fixed and stained with anti-myc. Scalebars = 20 µm. Quantification as in E. beclin 1-myc wt (29,1) vs. beclin 1ΔCCD-myc (28,3) significantly different (p<0.0001) using Fisher's exact test (two-sided).

Article Snippet: Immunoprecipitation was performed with 500 μg protein from post-nuclear supernatant, using antibodies against VPS34 (Echelon), UVRAG (Bethyl), or ATG14 (MBL) overnight.

Techniques: Dispersion, Over Expression, Staining, Mutagenesis, Transfection, Binding Assay

Journal: Nature Communications

Article Title: Class III PI3K regulates organismal glucose homeostasis by providing negative feedback on hepatic insulin signalling

doi: 10.1038/ncomms9283

Figure Lengend Snippet: ( a ) ATG14- and UVRAG-containing Vps34 complexes were immunoprecipitated from Hepa1.6 cells grown in the presence (NR) or absence of amino acids (−AA) and assayed for lipid kinase activity. Inputs for each assay were immunoblotted to determine the amounts of the Vps34 co-immunoprecipitated. PI3P signals were densitometrically measured and normalized to Vps34 protein levels. The data are presented as a fold difference of PI3P normalized to co-immunoprecipitated Vps34 levels revealed by immunoblot for each condition. Data are means±s.e.m. ( n =4, * P <0.05 versus NR, two-tailed, unpaired Student's t -test). ( b ) ATG14- and UVRAG-containing Vps34 complexes were immunoprecipitated from primary hepatocytes, which were serum starved for 24 h followed by stimulation with 1 μM insulin (Ins) and assayed for lipid kinase activity. PI3P signals were densitometrically measured. The data are presented as a fold difference of PI3P normalized to co-immunoprecipitated Vps34 levels revealed by immunoblot for each condition. Data are means±s.e.m. ( n =3, * P <0.05 versus starved cells, two-tailed, unpaired Student's t -test). Representative immunoblot analyses of ATG14, UVRAG and Vps34 co-immunoprecipitates are presented as and . ( c ) Primary hepatocytes were transiently transfected with UVRAG–Flag cDNA-expressing vector 12 h post-plating and were kept in the serum containing media. The 36-h post-transfection cells were collected and UVRAG–Flag complexes were immunoprecipitated with anti-Flag antibody. The presence of IRβ and class III PI3K subunits in the immunoprecipitation eluates was revealed by immunoblot. ( d ) Endogenous IRβ was immunoprecipitated from primary hepatocytes, which were serum starved for 24 h and stimulated with 1 μM insulin. The presence of Vps15 and IRβ in the immunoprecipitation eluates was revealed by immunoblot. Densitometric analyses of co-immunoprecipitated endogenous Vps15 normalized to IRβ are presented as fold difference over the unstimulated condition. Data are means±s.e.m. ( n =3, * P <0.05 versus starved cells, two-tailed, unpaired Student's t -test). ( e ) UVRAG-containing complexes were immunoprecipitated from primary hepatocytes, which were serum starved for 24 h followed by stimulation with 1 μM insulin for indicated times. Immunoprecipitated endogenous class III PI3K subunits were revealed by immunoblotting. Densitometric analyses of co-immunoprecipitated endogenous Rubicon normalized to UVRAG are presented as fold difference over the unstimulated condition. Data are means±s.e.m. ( n =3, * P <0.05 versus starved cells, two-tailed, unpaired Student's t -test). The protein G beads served as a control of the nonspecific binding in c – e .

Article Snippet: For class III PI3K in vitro activity antibodies were from MBL International ATG14 (PD026) and UVRAG (M160-3).

Techniques: Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Transfection, Expressing, Plasmid Preparation, Binding Assay