Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Silencing Enhances Autophagic Flux (A) Drosophila S2R + cells expressing GFP-LC3 were transfected with double-stranded RNA (dsRNA) targeting control luciferase (Luc) or Fs(1)h. (B and C) KP-4 cells transfected with control or BRD4 siRNA for 72 hr were subjected to western blot analysis (B) and stained for LC3B (C). The number of LC3 puncta normalized to cell number is shown. CON: n = 94 cells, BRD4 1: n = 97 cells, BRD4 2: n = 74 cells. Scale bars, 50 μm. (D) Immunohistochemistry of small intestinal sections from transgenic mice harboring inducible renilla luciferase or BRD4 shRNA. Sections were stained for LC3 (upper) and BRD4 (lower). Cytoplasmic signal in BRD4 panels is due to non-specific staining. Scale bars, 50 μm. (E) KP-4 cells transfected with BRD4 siRNA were treated with 10 μM CQ for 4 hr. (F) KP-4 cells transfected with BRD4 siRNA were stained for WIPI2. The number of WIPI2 puncta normalized to cell number is shown. CON: n = 119 cells, BRD4 1: n = 107 cells, BRD4 2: n = 109 cells. Scale bars, 20 μm. (G) KP-4 cells stably expressing RFP-GFP-LC3 were transfected with BRD4 siRNA. Scale bars, 50 μm. (H) KP-4 cells were treated with 500 nM JQ1 for 9 hr in the presence or absence of CQ (10 μM, 4 hr). (I) KP-4 cells overexpressing BRD4 were treated with 10 μM CQ for 4 hr. (J) TY-82 cells transfected with NUT siRNA for 5 days were treated with 10 μM CQ for 8 hr. BRD4-NUT was detected using NUT antibody. All data are shown as mean ± SD. ∗ p < 0.01. See also Figure S1 .

Article Snippet: Chloroquine (Cat#: C6628), Doxycycline (Cat#: D9891), Antimycin A (Cat#: A8674), Oligomycin (Cat#: O4876), D-(+)-Trehalose dihydrate (Cat#: T0167), and 4-Hydroxytamoxifen (Cat#: H7904) were from Sigma-Aldrich. (+)-JQ1 (Cat#: 4499) and I-BET151 (Cat#: 4650) were from TOCRIS.

Techniques: Expressing, Transfection, Control, Luciferase, Western Blot, Staining, Immunohistochemistry, Transgenic Assay, shRNA, Stable Transfection

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Is a Negative Regulator of Autophagy Gene Expression (A and B) KP-4 cells transfected with control or BRD4 siRNA were subjected to RNA-seq and gene ontology analyses (A) and RT-qPCR analysis (B). (C) RT-qPCR analysis of KP-4 cells treated with DMSO, 500 nM JQ1, 500 nM I-BET151, or 500 nM OTX015 for 9 hr. (D) RT-qPCR analysis of KP-4 cells treated with 500 nM JQ1 for the indicated time. (E) RT-qPCR analysis of KP-4 cells overexpressing BRD4. All data are shown as mean ± SD. In (A)–(D), n = 3 independent experiments; in (E), data are representative of two independent experiments performed in triplicate. ∗ p < 0.01, ∗∗ p < 0.05. See also Figure S2 .

Article Snippet: Chloroquine (Cat#: C6628), Doxycycline (Cat#: D9891), Antimycin A (Cat#: A8674), Oligomycin (Cat#: O4876), D-(+)-Trehalose dihydrate (Cat#: T0167), and 4-Hydroxytamoxifen (Cat#: H7904) were from Sigma-Aldrich. (+)-JQ1 (Cat#: 4499) and I-BET151 (Cat#: 4650) were from TOCRIS.

Techniques: Gene Expression, Transfection, Control, RNA Sequencing, Quantitative RT-PCR

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Knockdown Enhances Lysosomal Function (A) RT-qPCR analysis of KP-4 cells transfected with control or BRD4 siRNA. (B–D) KP-4 cells transfected with BRD4 siRNA were subjected to western blot analysis with antibodies against lysosomal proteins (B) and stained with LAMP1 antibody (C), LysoTracker Red (100 nM, 2 hr) (D, upper panels), and Magic Red CTSB (1 hr) (D, lower panels). Area of LAMP1 + , LysoTracker + , and Magic Red CTSB + area normalized to cell number is shown (C, CON: n = 115 cells, BRD4: n = 130 cells; D upper, CON: n = 66 cells, BRD4 1: n = 52 cells, BRD4 2: n = 50 cells; D lower, CON: n = 164 cells, BRD4 1: n = 109 cells, BRD4 2: n = 53 cells). Scale bars, 50 μm. (E) Hexosaminidase activity was measured using lysates from control and BRD4 knockdown KP-4 cells. (F and G) RT-qPCR analysis of TY-82 cells transfected with NUT siRNA for 72 hr (F) or treated with 500 nM JQ1 for 9 hr (G). (H) KP-4 cells were transfected with BRD4 and/or MiT/TFE (TFEB, TFE3, MITF) siRNAs and treated with 10 μM CQ for 4 hr. All data are shown as mean ± SD. In (A) and (F), n = 3 independent experiments. In (E), n = 4 independent experiments. In (G), data are representative of two independent experiments performed in triplicate. ∗ p < 0.01, ∗∗ p < 0.05. See also Figure S3 .

Article Snippet: Chloroquine (Cat#: C6628), Doxycycline (Cat#: D9891), Antimycin A (Cat#: A8674), Oligomycin (Cat#: O4876), D-(+)-Trehalose dihydrate (Cat#: T0167), and 4-Hydroxytamoxifen (Cat#: H7904) were from Sigma-Aldrich. (+)-JQ1 (Cat#: 4499) and I-BET151 (Cat#: 4650) were from TOCRIS.

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Control, Western Blot, Staining, Activity Assay

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet: BRD4 Represses Autophagy Gene Expression through G9a (A) Cell extracts from KP-4 cells were subjected to immunoprecipitation with G9a (upper) and BRD4 (lower) antibodies. (B) KP-4 cells were starved for 4 hr followed by immunoprecipitation with BRD4 antibody. (C–F) KP-4 cells were treated with 500 nM JQ1 for 9 hr (C and E). KP-4 cells harboring inducible control or BRD4 shRNA were treated with 500 ng/mL doxycycline (DOX) for 4 days (D and F). ChIP assays were performed using G9a (C and D) and H3K9diMe (E and F) antibodies. (G and H) KP-4 cells infected with shRNA targeting G9a were transfected with BRD4 siRNA followed by RT-qPCR (G) and western blot (H). (I) KP-4 cells overexpressing BRD4 were infected with shRNA targeting G9a. All data are shown as mean ± SD. In (C)–(G), n = 3 independent experiments. ∗ p < 0.01, ∗∗ p < 0.05, N.S., no significance. See also Figure S5 .

Article Snippet: Chloroquine (Cat#: C6628), Doxycycline (Cat#: D9891), Antimycin A (Cat#: A8674), Oligomycin (Cat#: O4876), D-(+)-Trehalose dihydrate (Cat#: T0167), and 4-Hydroxytamoxifen (Cat#: H7904) were from Sigma-Aldrich. (+)-JQ1 (Cat#: 4499) and I-BET151 (Cat#: 4650) were from TOCRIS.

Techniques: Gene Expression, Immunoprecipitation, Control, shRNA, Infection, Transfection, Quantitative RT-PCR, Western Blot

Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet:

Article Snippet: Chloroquine (Cat#: C6628), Doxycycline (Cat#: D9891), Antimycin A (Cat#: A8674), Oligomycin (Cat#: O4876), D-(+)-Trehalose dihydrate (Cat#: T0167), and 4-Hydroxytamoxifen (Cat#: H7904) were from Sigma-Aldrich. (+)-JQ1 (Cat#: 4499) and I-BET151 (Cat#: 4650) were from TOCRIS.

Techniques: Control, Membrane, Virus, Recombinant, Sample Prep, Flow Cytometry, Western Blot, Microscopy, Plasmid Preparation, Phospho-proteomics, Variant Assay, shRNA, Software, CRISPR

Journal: Medicina

Article Title: Ameliorative Effects of Cardamonin on Monosodium Urate-Induced Gouty Arthritis through Inhibiting NLRP3 Inflammasome Mediation

doi: 10.3390/medicina57090898

Figure Lengend Snippet: Effects of cardamonin on MSU-stimulated NLRP3 inflammasome activation in J774A.1 macrophage cells treated with MSU for 6 h in the presence or absence of cardamonin: ( A ) Representative gel showing the effect of cardamonin on MSU-stimulated cells. ( B ) Western blot analysis used to detect levels of NLRP3, caspase-1, and IL-1β in supernatants. (–) not treatment; (+) treatment. Each value represents the mean ± SD. Statistical differences among the MSU groups were compared against the control values, whereas cardamonin groups were compared against the MSU group using the ANOVA test ( n = 5; * p < 0.05 vs. control; # p < 0.05 vs. MSU; ## p < 0.01 vs. MSU).

Article Snippet: Specific antibodies were purchased from the following commercial suppliers: IL-1β, Catalog number: 4499 (Cell Signaling Technology, Danvers, MA, USA); NLRP-3, Catalog number: AG-20B-0014 (AdipoGen Life Sciences, San Diego, CA, USA); caspase-1, Catalog number: 4199 (Cell Signaling Technology, Danvers, MA, USA); COX-2, Catalog number: RB-9071 (Abcam, Cambridge, UK); and β-actin, Catalog number: sc-47778 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Western Blot, Control

Journal: Molecular cell

Article Title: Mutant p53 Gains Its Function via c-Myc Activation upon CDK4 Phosphorylation at Serine 249 and Consequent PIN1 Binding

doi: 10.1016/j.molcel.2017.11.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-p53 (DO-1) Santa Cruz Biotechnology Cat# sc-126 AB_628082 Mouse monoclonal anti-p53 (DO-1) Santa Cruz Biotechnology Cat# sc-126 X Rabbit polyclonal anti-p53 (FL-393) Santa Cruz Biotechnology Cat# sc-6243 AB_653753 Normal mouse IgG Santa Cruz Biotechnology Cat# sc-2025 AB_737182 Rabbit monoclonal anti-GAPDH Cell Signaling Technology Cat# 5174 AB_10622025 Rabbit monoclonal anti-Histone H3 Cell Signaling Technology Cat# 4499 AB_10544537 Mouse monoclonal anti-cdc2 (CDK1) Cell Signaling Technology Cat# 9116P AB_2074795 Rabbit monoclonal anti-CDK2 Cell Signaling Technology Cat# 2546P AB_2276129 Rabbit monoclonal anti-CDK4 Cell Signaling Technology Cat# 12790P AB_2631166 Rabbit monoclonal anti-CDK6 Cell Signaling Technology Cat# 13331P Mouse monoclonal anti-CDK7 Cell Signaling Technology Cat# 2916P AB_10827986 Rabbit monoclonal anti-CDK9 Cell Signaling Technology Cat# 2316P AB_2291505 Mouse monoclonal anti-CDK4 Santa Cruz Biotechnology Cat# sc-56277 AB_1121419 Rabbit polyclonal anti-Phosphorylation of S249 This paper N/A Mouse monoclonal anti-Flag M2 Sigma-Aldrich Cat# F3165 AB_259529 Mouse monoclonal anti-Cyclin D1 Santa Cruz Biotechnology Cat# sc-20044 AB_627346 Mouse monoclonal anti-Cyclin D3 Cell Signaling Technology Cat# 2936P AB_10841292 Rabbit polyclonal anti-Pin1 Cell Signaling Technology Cat# 3722S AB_10692654 Rabbit polyclonal anti-Pin1 Santa Cruz Biotechnology Cat# sc-15340 AB_2237080 Mouse monoclonal anti-Pin1 Santa Cruz Biotechnology Cat# sc-46660 AB_628132 Rabbit monoclonal anti-PARP Cell Signaling Technology Cat# 9532S AB_10695538 Rabbit monoclonal anti-c-Myc Abcam Cat# ab32072 AB_731658 Mouse monoclonal anti-GFP Santa Cruz Biotechnology Cat# sc-9996 AB_627695 Mouse monoclonal anti-P21 WAF1 NeoMarkers Cat#MS-891-P1 Mouse monoclonal anti-beta-Actin Sigma-Aldrich Cat# A1978 AB_476692 Mouse monoclonal anti-beta-Actin Santa Cruz Biotechnology Cat# sc-47778 AB_626632 Mouse monoclonal anti-alpha-tubulin Sigma-Aldrich Cat# 00020911 AB_10013740 Peroxidase AffiniPure Goat Anti-Mouse IgG, Light Chain Specific for Western blotting after IP Jackson ImmunoResearch Cat# 115-035-174 AB_2338512 Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific Jackson ImmunoResearch 211-032-171 AB_2339149 Mouse IgG Isotype Control Thermo Fisher Cat# 31903 AB_10959891 Rabbit IgG Isotype Control Thermo Fisher Cat# 31235 AB_243593 Bacterial and Virus Strains DH5a competent cells NEW ENGLAND BioLabs Cat# C2987H BL21(DE3) competent cells NEW ENGLAND BioLabs Cat# C2527H Stbl3 competent cells Thermo Fisher Cat# A10469 Biological Samples Hepatocellular carcinoma (HCC) tissue samples The First Affiliated Hospital of Nanchang University, Jiangxi, China.

Techniques: Western Blot, Control, Virus, Recombinant, CCK-8 Assay, Plasmid Preparation, Software

Journal: eLife

Article Title: GLI transcriptional repression regulates tissue-specific enhancer activity in response to Hedgehog signaling

doi: 10.7554/eLife.50670

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Histone H3 (rabbit polyclonal , Cell Signaling Technology , Cell Signaling Technology Cat# 4499, RRID: AB_10544537 , Used for WB (1:4000).

Techniques: MicroChIP Assay, Cell Culture, Software