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: BRD4 E2A7X (long isoform) (Cat#: 13440S, WB 1/1000, IP, ChIP), NUT (Cat#: 3625S, WB 1/1000), G9a (Cat#: 3306S, WB 1/1000), SIRT1 (Cat#: 9475P, WB 1/1000), DBC1 (Cat#: 5857S, WB 1/1000, IP), AMPKα pT172 (Cat#: 2535S, WB 1/1000), AMPKα1/α2 (Cat#: 2793S, WB 1/1000), LC3B (Cat#: 2775S, WB 1/1500), LC3B D11 (Cat#: 3868S, IF 1/200), TFEB (Cat#: 4240S, WB 1/1000), LAMP1 (Cat#: 9091P, WB 1/1000, IF 1/200), CTSD (Cat#: 2284S, WB 1/1000), ASM (Cat#: 3687S, WB 1/1000), CTSB (Cat#: 31718S, WB 1/1000), ATG5 (Cat#: 12994S, WB 1/1500), p70 S6K pT389 (Cat#: 9234S, WB 1/1000), p70 S6K (Cat#: 2708S, WB 1/1500), Histone H4K16Ac (Cat#: 13534S, WB 1/1000), Histone H4 (Cat#: 2935P, WB 1/1000), c-Myc (Cat#: 5605S, WB 1/1000), CDK9 (Cat#: 2316T, WB 1/1000), GFP (Cat#: 2956S, WB 1/2000), TFE3 (Cat#: 14779S, WB 1/1000), Normal rabbit IgG (Cat#: 2729S, IP, ChIP), Mouse IgG1 isotype control (Cat#: 5415S, IP), Anti-rabbit IgG HRP-linked Antibody (Cat#: 7074S, WB 1/5000), and Anti-mouse IgG HRP-linked Antibody (Cat#: #7076S, WB 1/5000) were from Cell Signaling Technology.

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:

Article Snippet: BRD4 E2A7X (long isoform) (Cat#: 13440S, WB 1/1000, IP, ChIP), NUT (Cat#: 3625S, WB 1/1000), G9a (Cat#: 3306S, WB 1/1000), SIRT1 (Cat#: 9475P, WB 1/1000), DBC1 (Cat#: 5857S, WB 1/1000, IP), AMPKα pT172 (Cat#: 2535S, WB 1/1000), AMPKα1/α2 (Cat#: 2793S, WB 1/1000), LC3B (Cat#: 2775S, WB 1/1500), LC3B D11 (Cat#: 3868S, IF 1/200), TFEB (Cat#: 4240S, WB 1/1000), LAMP1 (Cat#: 9091P, WB 1/1000, IF 1/200), CTSD (Cat#: 2284S, WB 1/1000), ASM (Cat#: 3687S, WB 1/1000), CTSB (Cat#: 31718S, WB 1/1000), ATG5 (Cat#: 12994S, WB 1/1500), p70 S6K pT389 (Cat#: 9234S, WB 1/1000), p70 S6K (Cat#: 2708S, WB 1/1500), Histone H4K16Ac (Cat#: 13534S, WB 1/1000), Histone H4 (Cat#: 2935P, WB 1/1000), c-Myc (Cat#: 5605S, WB 1/1000), CDK9 (Cat#: 2316T, WB 1/1000), GFP (Cat#: 2956S, WB 1/2000), TFE3 (Cat#: 14779S, WB 1/1000), Normal rabbit IgG (Cat#: 2729S, IP, ChIP), Mouse IgG1 isotype control (Cat#: 5415S, IP), Anti-rabbit IgG HRP-linked Antibody (Cat#: 7074S, WB 1/5000), and Anti-mouse IgG HRP-linked Antibody (Cat#: #7076S, WB 1/5000) were from Cell Signaling Technology.

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

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: NEDD4L mediated ubiquitylation and degradation of YAP1 through binding to YAP1. (A) MDA‐MB‐231 parent cell lysates were immunoprecipitated with anti‐YAP1 antibody. The red arrow indicates the YAP1‐interacting protein identified by mass spectrometry. (B) The E3 ubiquitin ligases acting on YAP1 were predicted with the online database UbiBrowser, an integrated bioinformatics platform. The eight dots in red refer to those verified as E3 ubiquitin ligases of YAP1 in the literatures, and the 10 dots in blue are predicted as E3 ubiquitin ligases of YAP1. The capital letters in dots indicate the initial letters of E3 ubiquitin ligases‐domains: F refers to F‐box domain, R refers to RING domain, H refers to HECT domain, U refers to UBOX domain. The predicted interactions are arranged in descending order clockwise based on the confidence score. (C) NEDD4L and YAP1 were immunoprecipitated from MCF7 and SKBR3 parent cell lysates with anti‐YAP1 and anti‐NEDD4L antibodies, then detected via WB. This confirmed the interaction between YAP1 and NEDD4L. (D) Representative IHC staining images of NEDD4L and YAP1 in breast cancer tissues ( n = 51) (left panel). Expression levels of NEDD4L and YAP1 were negatively correlated with one another (right panel). (E) WB analysis of NEDD4L expression in six matched pairs of primary breast cancer tissues (T) and adjacent normal tissues (N). (F) MCF7 and SKBR3 cells were transfected with NEDD4L ‐ siRNAs ( siNEDD4L#1 , siNEDD4L#2 , siNEDD4L#3 ), and NEDD4L expression was detected with WB analysis. siNEDD4L#3 was selected for subsequent experiments. (G) Stability analysis of YAP1. MCF7 and SKBR3 cells were transfected with NEDD4L overexpression plasmid or siNEDD4L for 48 h, then exposed to CHX (100 µg/mL) for 0, 8, 10, or 12 h. WB analysis of YAP1 expression was then performed. (H‐I) YAP1 ubiquitination degradation assay. MCF7 and SKBR3 cells were transfected with NEDD4L overexpression plasmid or siNEDD4L and treated with MG132 (20 µmol/L, 6 h). YAP1 expression was detected with WB analysis. (J) Ubiquitination assays showing the effects of NEDD4L on YAP1 ubiquitination. MCF7 and SKBR3 cells overexpressing Flag‐ub were transfected with NEDD4L overexpression plasmid or siNEDD4L then treated with MG132 (20 µmol/L, 6 h). WB analysis was used to detect the ubiquitination of YAP1. Each experiment was repeated three times. Abbreviations: NEDD4L, NEDD4 like E3 ubiquitin protein ligase; IP, immunoprecipitation; IgG, immunoglobulin G; WB, Western blotting; YAP1, Yes1‐associated transcriptional regulator; IHC, immunohistochemistry; CHX, cycloheximide; Ub, ubiquitin.

Article Snippet: Antibodies for YAP1 (13584‐1‐AP, 66900‐1‐Ig; 1:1000 for WB, 1:200 for immunohistochemistry [IHC], 1:100 for IF), NEDD4 like E3 ubiquitin protein ligase (NEDD4L, 13690‐1‐AP, 1:1000 for WB, 1:400 for IHC), sequestosome 1 (SQSTM1/p62, 18420‐1‐AP, 1:1000 for WB, 1:50 for IHC) and CYR61 (26689‐1‐AP, 1:1000 for WB, 1:50 for IHC) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Binding Assay, Immunoprecipitation, Mass Spectrometry, Ubiquitin Proteomics, Immunohistochemistry, Expressing, Transfection, Over Expression, Plasmid Preparation, Degradation Assay, Western Blot

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: The mechanism of cytoplasmic YAP1‐mediated ESCRT‐III assembly promoting autophagic cell death. Abbreviations: YAP1, Yes1‐associated transcriptional regulator; ESCRT, endosomal sorting complexes required for transport; EGCG, epigallocatechin gallate; NEDD4L, NEDD4 like E3 ubiquitin protein ligase; CHMP2B, charged multivesicular body protein 2B; VPS4B, vacuolar protein sorting 4 homolog B; LC3, microtubule associated protein 1 light chain 3; Ub, ubiquitin.

Article Snippet: Antibodies for YAP1 (13584‐1‐AP, 66900‐1‐Ig; 1:1000 for WB, 1:200 for immunohistochemistry [IHC], 1:100 for IF), NEDD4 like E3 ubiquitin protein ligase (NEDD4L, 13690‐1‐AP, 1:1000 for WB, 1:400 for IHC), sequestosome 1 (SQSTM1/p62, 18420‐1‐AP, 1:1000 for WB, 1:50 for IHC) and CYR61 (26689‐1‐AP, 1:1000 for WB, 1:50 for IHC) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Ubiquitin Proteomics