Journal: Journal of medicinal chemistry

Article Title: Structure-Guided Design of ISOX-DUAL-Based Degraders Targeting BRD4 and CBP/EP300: A Case of Degrader Collapse.

doi: 10.1021/acs.jmedchem.5c00395

Figure Lengend Snippet: Figure 3. In vitro ubiquitination assays for CRBN-recruiting degraders 34−36 with BRD4 (left panel) and CBP (right panel). FLAG-BRD4 and GST- CREBBP were detected using capillary electrophoresis (Simple Western, Wes) and anti-FLAG and anti-GST antibodies.

Article Snippet: 50 μL in vitro reactions included the following components and concentrations: UBE1 (E-304−050, R&D Systems), 100 nM; UBE2D1 (E2−616, R&D Systems), 1 μM; CRBN/ DDB1/CUL4A(neddylated)/RBX1 complex (E3−441 and E3−500, premixed), 50 nM; FLAG-BRD4 (residues 49−460, SP-600, R&D Systems), 2 μM; ubiquitin (U-100H, R&D Systems), 50 μM; ATP (B20, R&D Systems), 10 mM; degrader compound, variable concentrations.

Techniques: In Vitro, Ubiquitin Proteomics, Electrophoresis, Simple Western

Journal: Pathology and Oncology Research

Article Title: Autophagy Plays a Role in the CUL4A-Related Poor Prognosis of Intrahepatic Cholangiocarcinoma

doi: 10.3389/pore.2021.602714

Figure Lengend Snippet: Clinicopathological characteristics and association with autophagy of patients with intrahepatic cholangiocarcinoma.

Article Snippet: The lentivirus vectors carrying CUL4A were purchased from OriGene (OriGene, MD, United States).

Techniques: Over Expression

Journal: Pathology and Oncology Research

Article Title: Autophagy Plays a Role in the CUL4A-Related Poor Prognosis of Intrahepatic Cholangiocarcinoma

doi: 10.3389/pore.2021.602714

Figure Lengend Snippet: Comparisons of autophagic flux between iCCA cells with or without CUL4A overexpression. Cells with CUL4A overexpression showed a much greater accumulation of LC3II in CUL4A overexpression cells after bafilomycin A1 treatment. (A) The iCCA cells were treated with the presence or absence of 100 nm bafilomycin A1 and examined LC3 expression in the course of the indicated time point. The levels of LC3II expression are examined by Immunoblotting followed by quantitative analysis using Image J . Data are normalized to GAPDH level. (B) Values indicative of autophagic flux are quantitatively determined by expression difference of LC3II amount between cells treated with DMSO and cells treated with bafilomycin A1. The scale represents ratio of LC3II following bafilomycin A1 treatment/untreated. Data represent means ± standard deviation from three independent experiments. **, p < 0.01.

Article Snippet: The lentivirus vectors carrying CUL4A were purchased from OriGene (OriGene, MD, United States).

Techniques: Over Expression, Expressing, Western Blot, Standard Deviation

Journal: Cell Death & Disease

Article Title: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism

doi: 10.1038/s41419-022-04798-6

Figure Lengend Snippet: A Lentiviral shRNA knockdown of CUL4A decreases DDB2 expression. FaDu cells were transfected with lentiviral shRNA particles targeting CUL4A, CUL4B or a scramble control. The levels of CUL4A, CUL4B, and DDB2 were determined by immunoblotting. B Knockdown of CUL4A leads to increased γH2AX expression. FaDu cells were transfected with lentiviral shRNA targeting CUL4A, CUL4B, or a scramble control. Cells were then treated with 3 μM cisplatin for 24 h. γH2AX levels were determined and quantified by ImageJ software. DAPI was used as a counterstain. Representative images are shown. Mean signal intensity ±SEM, n = 20 cells per condition. **Indicates a significant difference from other treated groups, p < 0.01. C FaDu cells were transfected with lentiviral particles containing a functional CUL4A expression vector. Overexpression of CUL4A was confirmed by immunoblotting following puromycin selection. D Overexpression of CUL4A decreases cisplatin-mediated DNA damage. FaDu cells transfected with CUL4A overexpression and control plasmid were incubated with 5 μM cisplatin for 24 h. γH2AX expression was observed and quantified using ImageJ software. DAPI was used as a nuclear counterstain. Representative images are shown. Mean signal intensity ±SEM, n = 66 cells per condition. Scale bar = 20 μm. **Indicates a significant difference between samples, p < 0.01.

Article Snippet: FaDu cells were infected with lentiviral particles containing a puromycin resistance gene, a CUL4A (#RC214798L4V) cDNA construct, or a DDB2 (#RC200390L4V) cDNA construct according to the manufacturer’s protocol (Origene, Rockville, MD).

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, Western Blot, Software, Functional Assay, Plasmid Preparation, Over Expression, Selection, Incubation