Journal: Scientific Reports

Article Title: Identification of highly potent and selective inhibitor, TIPTP, of the p22phox-Rubicon axis as a therapeutic agent for rheumatoid arthritis

doi: 10.1038/s41598-020-61630-x

Figure Lengend Snippet: p22phox interaction with Rubicon in human and CIA mice. ( a ) Schematic of the collagen-induced RA (CIA) model (upper). p22phox complexes purified from synovial fluid containing synoviocytes from CIA mice were subjected to mass spectrometry analysis. Silver stained gel (bottom left) and peptides identified by mass spectrometry analysis (bottom right) The red-colored letters indicate the peptides identified from mass spectrometry analysis. ( b ) Representative Hematoxylin-Eosin (H&E) staining of the ankle joints of each group (upper); Scale bar, 500 μm. Clinical arthritis, swelling of paws scores (middle), and histopathology scores (bottom). Results are expressed as means ± SD (5 mice per group). ( c ) Synovial fluid containing synoviocytes from CIA mice for the indicated times, followed by IP with αp22phox (left) or αRubicon (right), followed by IB with αRubicon, αp22phox, and αgp91phox. The anti-p22phox blot was also tested for αTLR4, αTRAF6 binding. The anti-Rubicon blot was also tested for αBeclin-1, or αUVRAG. WCLs were used for IB with αRubicon, αp22phox, αgp91phox, αTLR4, αTRAF6, αBeclin-1, αUVRAG. Actin Western blot was used as a loading control. ( d ) OA and RA cells were stained with αp22phox (Alexa Fluor 488; green) and αRubicon (Cy3; red). Nuclei were counterstained with DAPI. Cells were visualized by confocal microscopy (left). The middle panel shows the quantitative data of staining intensity of p22phox and Rubicon (upper) and the colocalization index (%) between p22phox and Rubicon (bottom). Immunohistochemical analysis to examine p22phox-DAB (3,3′-diaminobenzidine) and Rubicon-AEC (3-amino-9-ethylcarbazole) expression (right). Representative images from six independent OA and RA patients are shown. Insets, enlargement of outlined areas. Scale bars: 200 μm. The data are representative of five independent experiments with similar results ( a , c and d ). Statistical analysis was done using the Student’s t -test with Bonferroni adjustment. Data are considered different at p < 0.05. ***p < 0.001. Experimental procedures were described in Supplementary Information.

Article Snippet: Antibodies against Beclin-1 (3738) and UVRAG (5320) were purchased from Cell Signaling Technology.

Techniques: Purification, Mass Spectrometry, Staining, Histopathology, Binding Assay, Western Blot, Control, Confocal Microscopy, Immunohistochemical staining, Expressing

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