Journal: ACS chemical biology

Article Title: High Throughput Screens to Identify Autophagy Inducers that Function by Disrupting Beclin 1/Bcl-2 Binding

doi: 10.1021/acschembio.8b00421

Figure Lengend Snippet: Development of Beclin 1/Bcl-2 split-luciferase assay. (A) Schematic diagram showing constructs used in Beclin 1/Bcl-2 split-luciferase assay. (B) Split-luciferase activity measuring the Beclin 1 and Bcl-2 (black bars) or Beclin 1ΔBcl−2BD and Bcl-2 (gray bars) interaction in the presence of increasing concentrations of the BH3 mimetic compound, ABT-737 (treatment duration, 4 h). Results are shown as normalized relative luminescence units (RLU), which is the ratio of split-luciferase activity to Renilla luciferase control activity. The value for Beclin 1/Bcl-2 interaction in the absence of ABT-737 was arbitrarily set as 100%. Results shown are mean ± s.d. from a representative experiment. ***p<0.001; Student’s t-test. Similar results were obtained in three independent experiments. (C) Western blot analysis of NLuc-Beclin 1 and CLuc-Bcl-2 expression with anti-luciferase antibody in cell lysates used in experiment shown in (B). (D) Uniformity assay showing Z’ factor test to validate the Beclin 1/Bcl-2 split-luciferase in a 384-well plate HTS format. DMSO (gray circles, 240 wells) or ABT-737 (white circles, 16 wells) were used as neutral or positive controls, respectively.

Article Snippet: The following antibodies were used for western blot analyses: goat polyclonal anti-luciferase (Promega G7451, 1:1000 dilution), rabbit anti-Beclin 1 (Santa Cruz sc-11427, 1:500 dilution), HRP-conjugated anti-Myc (Santa Cruz SC-40 HRP, 1:100 dilution), rabbit anti-Bax (Cell Signaling Technology #5023, 1:500 dilution), rabbit anti-LC3 (Novus NB100–2220, 1:2000 dilution), mouse anti-p62 (Abnova #H00008878-M01, 1:20000 dilution), rabbit anti-Bim (Cell Signaling Technology #2933, 1:1000 dilution), rabbit anti-cleaved PARP (Cell Signaling Technology #9451, 1:1000 dilution) and HRP-conjugated anti-actin (Santa Cruz sc-47778-HRP, 1:5000 dilution).

Techniques: Luciferase, Construct, Activity Assay, Western Blot, Expressing

Journal: ACS chemical biology

Article Title: High Throughput Screens to Identify Autophagy Inducers that Function by Disrupting Beclin 1/Bcl-2 Binding

doi: 10.1021/acschembio.8b00421

Figure Lengend Snippet: Development of Beclin 1/Bcl-2 AlphaLISA assay. (A) Schematic diagram showing constructs used in Beclin 1/Bcl-2 AlphaLISA assay. (B) AlphaLISA assay activity measuring the Beclin 1 and Bcl-2 (black bars) or Beclin 1ΔBcl−2BD and Bcl-2 (gray bars) interaction in the presence of increasing concentrations of ABT-737 (treatment duration, 4 h). Results shown are mean ± s.d. from a representative experiment. **p<0.05; **p<0.01; ***p<0.001; Student’s t-test. Similar results were obtained in three independent experiments. (C) Uniformity assay showing Z’ factor test to validate the Beclin 1/Bcl-2 AlphaLISA asay in a 384-well plate HTS format. DMSO (gray circles, 240 wells) or ABT-737 (white circles, 16 wells) were used as neutral or positive controls, respectively.

Article Snippet: The following antibodies were used for western blot analyses: goat polyclonal anti-luciferase (Promega G7451, 1:1000 dilution), rabbit anti-Beclin 1 (Santa Cruz sc-11427, 1:500 dilution), HRP-conjugated anti-Myc (Santa Cruz SC-40 HRP, 1:100 dilution), rabbit anti-Bax (Cell Signaling Technology #5023, 1:500 dilution), rabbit anti-LC3 (Novus NB100–2220, 1:2000 dilution), mouse anti-p62 (Abnova #H00008878-M01, 1:20000 dilution), rabbit anti-Bim (Cell Signaling Technology #2933, 1:1000 dilution), rabbit anti-cleaved PARP (Cell Signaling Technology #9451, 1:1000 dilution) and HRP-conjugated anti-actin (Santa Cruz sc-47778-HRP, 1:5000 dilution).

Techniques: Construct, Activity Assay

Journal: ACS chemical biology

Article Title: High Throughput Screens to Identify Autophagy Inducers that Function by Disrupting Beclin 1/Bcl-2 Binding

doi: 10.1021/acschembio.8b00421

Figure Lengend Snippet: HTS algorithm for identifying compounds that disrupt Beclin 1/Bcl-2 interaction. Compounds that demonstrated Z-score ≤ −3.0 in primary and confirmation Beclin 1/Bcl-2 split-luciferase HTS screens were considered as initial hits. A selection of cherry-picked compounds (1027 from UTSW library and 55 from Broad library) was subjected to secondary screening using the Beclin 1/Bcl-2 AlphLISA HTS assay. In the secondary screen, compounds that demonstrated >20% (UTSW library) or >40% (Broad library) inhibition with a Z-score ≤ −3.0 were further analyzed in dose-response studies. A pan assay interference compound (PAINS) filter was applied to remove potential promiscuous inhibitors.

Article Snippet: The following antibodies were used for western blot analyses: goat polyclonal anti-luciferase (Promega G7451, 1:1000 dilution), rabbit anti-Beclin 1 (Santa Cruz sc-11427, 1:500 dilution), HRP-conjugated anti-Myc (Santa Cruz SC-40 HRP, 1:100 dilution), rabbit anti-Bax (Cell Signaling Technology #5023, 1:500 dilution), rabbit anti-LC3 (Novus NB100–2220, 1:2000 dilution), mouse anti-p62 (Abnova #H00008878-M01, 1:20000 dilution), rabbit anti-Bim (Cell Signaling Technology #2933, 1:1000 dilution), rabbit anti-cleaved PARP (Cell Signaling Technology #9451, 1:1000 dilution) and HRP-conjugated anti-actin (Santa Cruz sc-47778-HRP, 1:5000 dilution).

Techniques: Luciferase, Selection, HTS Assay, Inhibition, Pan Assay

Journal: ACS chemical biology

Article Title: High Throughput Screens to Identify Autophagy Inducers that Function by Disrupting Beclin 1/Bcl-2 Binding

doi: 10.1021/acschembio.8b00421

Figure Lengend Snippet: Hit validation and assessment of selectivity. (A) Structure of three compounds chosen for further validation and biological assays. (B) Dose-response curves of the effects of SW063058, SW076956 and BRD1991 on inhibition of Beclin 1/Bcl-2 interaction. Serially-diluted compounds (2-fold, 7 points) were tested simultaneously in the Beclin 1/Bcl-2, Beclin 1 BH3 peptide/Bcl-2, or Bax BH3 peptide/Bcl-2 AlphaLISA. Results shown are mean ± s.d. from a representative experiment. Similar results were observed in three independent experiments. p-values were calculated by pairwise comparisons to DMSO control. *p<0.05; **p<0.01; ***p<0.001; Student’s t-test. (C) Co-immunoprecipitation of Beclin 1, Bax or Bim with Myc-Bcl-2 in HeLa/Myc-Bcl-2 cells treated with 20 μM indicated compound for 12 h prior to analysis. Immunoprecipitation was performed with an anti-Myc antibody followed by western blot analysis of indicated protein.

Article Snippet: The following antibodies were used for western blot analyses: goat polyclonal anti-luciferase (Promega G7451, 1:1000 dilution), rabbit anti-Beclin 1 (Santa Cruz sc-11427, 1:500 dilution), HRP-conjugated anti-Myc (Santa Cruz SC-40 HRP, 1:100 dilution), rabbit anti-Bax (Cell Signaling Technology #5023, 1:500 dilution), rabbit anti-LC3 (Novus NB100–2220, 1:2000 dilution), mouse anti-p62 (Abnova #H00008878-M01, 1:20000 dilution), rabbit anti-Bim (Cell Signaling Technology #2933, 1:1000 dilution), rabbit anti-cleaved PARP (Cell Signaling Technology #9451, 1:1000 dilution) and HRP-conjugated anti-actin (Santa Cruz sc-47778-HRP, 1:5000 dilution).

Techniques: Inhibition, Immunoprecipitation, Western Blot

Journal: ACS chemical biology

Article Title: High Throughput Screens to Identify Autophagy Inducers that Function by Disrupting Beclin 1/Bcl-2 Binding

doi: 10.1021/acschembio.8b00421

Figure Lengend Snippet: Effects of Beclin 1/Bcl-2 binding disruptors on autophagy and cell death. (A-B) Representative images (A) or quantitation (B) of GFP-LC3-positive puncta in HeLa/GFP-LC3 cells treated with DMSO or indicated compound. 20 μM compound +/− 100 nM Baf A1, 24 h. Bars represent mean ± s.e.m. of triplicate samples (>100 cells analyzed per sample). Similar results were observed in three independent experiments. ***p<0.001; Mann–Whitney U test. Scale bars, 1 μm. (C) Western blot analysis of LC3 in HeLa cells treated with indicated concentration of indicated compound for 24 h in the presence or absence of 100 nM Baf A1. (D) Effects of indicated compounds at indicated concentrations on HeLa cell apoptosis as assessed by western blot detection of cleaved PARP.

Article Snippet: The following antibodies were used for western blot analyses: goat polyclonal anti-luciferase (Promega G7451, 1:1000 dilution), rabbit anti-Beclin 1 (Santa Cruz sc-11427, 1:500 dilution), HRP-conjugated anti-Myc (Santa Cruz SC-40 HRP, 1:100 dilution), rabbit anti-Bax (Cell Signaling Technology #5023, 1:500 dilution), rabbit anti-LC3 (Novus NB100–2220, 1:2000 dilution), mouse anti-p62 (Abnova #H00008878-M01, 1:20000 dilution), rabbit anti-Bim (Cell Signaling Technology #2933, 1:1000 dilution), rabbit anti-cleaved PARP (Cell Signaling Technology #9451, 1:1000 dilution) and HRP-conjugated anti-actin (Santa Cruz sc-47778-HRP, 1:5000 dilution).

Techniques: Binding Assay, Quantitation Assay, MANN-WHITNEY, Western Blot, Concentration Assay

Journal: Antioxidants

Article Title: Selected Soybean Varieties Regulate Hepatic LDL-Cholesterol Homeostasis Depending on Their Glycinin:β-Conglycinin Ratio

doi: 10.3390/antiox12010020

Figure Lengend Snippet: Pearson correlations between the concentration of glycinin and β-conglycinin, and the glycinin: β-conglycinin ratio and the evaluated biochemical and cell culture markers of cholesterol metabolism and LDL oxidation.

Article Snippet: Membranes were incubated overnight at 4 °C with primary mouse antibodies for human p-AMPK T172 (2535, Cell Signaling, Danvers, MA; epitope in T172), AMPK (sc-74461; epitope in amino acids 251–550), SREBP-2 (sc-271616, epitope in amino acids 812–975), HMGCR (sc-271595, epitope in amino acids 589–888), SIRT1 (sc-74504, epitope in amino acids 448–747), p-ACC S78/S80 (sc-271965 epitope in amino acids S78 and S80), ACC (sc-137104, epitope in amino acids 1–76), SREBP-1c (sc-17755, epitope in amino acids 41–200), FASN (sc-48357, epitope in amino acids 2205–2504), LDLR (sc-18823, epitope in amino acids 13–47), and PCSK9 (sc-515082, epitope in amino acids 175–334) purchased in Santa Cruz Biotechnology, unless otherwise stated.

Techniques: Concentration Assay, Cell Culture, Phospho-proteomics, Expressing, Activity Assay

Journal: Antioxidants

Article Title: Selected Soybean Varieties Regulate Hepatic LDL-Cholesterol Homeostasis Depending on Their Glycinin:β-Conglycinin Ratio

doi: 10.3390/antiox12010020

Figure Lengend Snippet: Effect of selected soybean digests (V1, V3, V9, V17, and V18 at 253, 160, 76, 64, and 146 µg protein mL −1 , respectively) and simvastatin (ST, 160 ng mL −1 ) on the relative protein expression/phosphorylation ( A ) of AMP-activated protein kinase (AMPK) as p-AMPK/AMPK ratio ( B ), sterol regulatory element-binding protein 2 (SREBP-2) ( C ), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) ( D ), HMGCR relative activity ( E ), and the intracellular concentration of free ( F ), esterified ( G ), and total ( H ) cholesterol and the release of apolipoprotein B (ApoB) ( I ) in free fatty-acid-stimulated HepG2 liver cells (500 µmol L −1 oleic: palmitic acid, 2:1). Results are reported as mean ± SD ( n = 3). Bars with different letters significantly differ according to ANOVA and Tukey’s multiple range test ( p < 0.05).

Article Snippet: Membranes were incubated overnight at 4 °C with primary mouse antibodies for human p-AMPK T172 (2535, Cell Signaling, Danvers, MA; epitope in T172), AMPK (sc-74461; epitope in amino acids 251–550), SREBP-2 (sc-271616, epitope in amino acids 812–975), HMGCR (sc-271595, epitope in amino acids 589–888), SIRT1 (sc-74504, epitope in amino acids 448–747), p-ACC S78/S80 (sc-271965 epitope in amino acids S78 and S80), ACC (sc-137104, epitope in amino acids 1–76), SREBP-1c (sc-17755, epitope in amino acids 41–200), FASN (sc-48357, epitope in amino acids 2205–2504), LDLR (sc-18823, epitope in amino acids 13–47), and PCSK9 (sc-515082, epitope in amino acids 175–334) purchased in Santa Cruz Biotechnology, unless otherwise stated.

Techniques: Expressing, Phospho-proteomics, Binding Assay, Activity Assay, Concentration Assay

Journal: Antioxidants

Article Title: Selected Soybean Varieties Regulate Hepatic LDL-Cholesterol Homeostasis Depending on Their Glycinin:β-Conglycinin Ratio

doi: 10.3390/antiox12010020

Figure Lengend Snippet: Effect of selected soybean digests (V1, V3, V9, V17, and V18 at 253, 160, 76, 64, and 146 µg protein mL −1 , respectively) and simvastatin (ST, 160 ng mL −1 ) on the intracellular lipid accumulation ( A , B ), triglyceride concentration ( C ), the relative protein expression/phosphorylation ( D ) of sirtuin 1 (SIRT1) ( E ), sterol regulatory element-binding protein 1c (SREBP-1c) ( F ), acetyl-CoA carboxylase (ACC) ( G ), and fatty acid synthase (FASN) ( H ) in free fatty-acid-stimulated HepG2 liver cells (500 µmol L −1 oleic: palmitic acid, 2:1). Results are reported as mean ± SD ( n = 3). Bars with different letters significantly differ according to ANOVA and Tukey’s multiple range test ( p < 0.05).

Article Snippet: Membranes were incubated overnight at 4 °C with primary mouse antibodies for human p-AMPK T172 (2535, Cell Signaling, Danvers, MA; epitope in T172), AMPK (sc-74461; epitope in amino acids 251–550), SREBP-2 (sc-271616, epitope in amino acids 812–975), HMGCR (sc-271595, epitope in amino acids 589–888), SIRT1 (sc-74504, epitope in amino acids 448–747), p-ACC S78/S80 (sc-271965 epitope in amino acids S78 and S80), ACC (sc-137104, epitope in amino acids 1–76), SREBP-1c (sc-17755, epitope in amino acids 41–200), FASN (sc-48357, epitope in amino acids 2205–2504), LDLR (sc-18823, epitope in amino acids 13–47), and PCSK9 (sc-515082, epitope in amino acids 175–334) purchased in Santa Cruz Biotechnology, unless otherwise stated.

Techniques: Concentration Assay, Expressing, Phospho-proteomics, Binding Assay

Journal: Journal of Neuroscience Research

Article Title: Neuronal extracellular microRNAs miR‐124 and miR‐9 mediate cell–cell communication between neurons and microglia

doi: 10.1002/jnr.24344

Figure Lengend Snippet: Antibody characterization

Article Snippet: Rabbit polyclonal antibodies for human/mouse/rat HDLs (immunogen: KLH conjugated synthetic peptide from a 41–125 amino acid sequence of human HDL) were purchased from Biorbyt (cat#orb1003; 0.5 mg/ml, dilution 1:1,000; RRID: AB_2737031).

Techniques: Concentration Assay, Marker, Binding Assay, Immunofluorescence, Blocking Assay, Sequencing, Western Blot