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: Fig. 4 Pevonedistat-mediated downregulation of DDB2 drives its synergy with cisplatin. A, B Pevonedistat decreases the expression of DDB2. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat (A) and were treated with 600 nM pevonedistat, 3 μM cisplatin, and the combination (B) for 24 h. DDB2 expression was determined by immunoblotting. C DDB2 was silenced in FaDu cells using lentiviral shRNA. Cells were infected with DDB2 and scramble control shRNA and placed under puromycin selection. DDB2 levels were determined by immunoblotting. D Knockdown of DDB2 increases γH2AX levels following cisplatin treatment. FaDu cells transfected with lentiviral shDDB2 or control particles were treated with 3 μM cisplatin for 24 h. γH2AX signal was observed by fluorescent imaging and quantified using ImageJ software. DAPI was used as a counterstain. Mean signal intensity ±SEM, n = 58 cells per condition. E Knockdown of DDB2 increases cisplatin-induced apoptosis. FaDu cells transfected with lentiviral DDB2 or control shRNA were treated with the indicated concentrations of cisplatin for 48 h. Apoptosis was determined by PI-FACS analysis. Mean ± SD, n = 3. **Indicates a significant difference between indicated groups, 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: Expressing, Western Blot, shRNA, Infection, Control, Selection, Knockdown, Transfection, Imaging, Software

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: Fig. 5 Inhibition of E2F1 underlies pevonedistat-driven suppression of DDB2 levels. A Pevonedistat treatment results in the transcriptional downregulation of DDB2. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat for 24 h. qRT-PCR was used to quantify changes in DDB2 expression. Mean ± SD, n = 3. B Pevonedistat decreases DDB2 promoter activity. FaDu cells were transfected with lentiviral particles containing a DDB2 promoter flanking a luciferase gene. Cells stably expressing this construct were treated with the indicated concentrations of pevonedistat for 24 h and DDB2 promoter activity was quantified. Mean ± SD, n = 3. *Indicates a significant difference from control, p < 0.05. C Pevonedistat increases p21 expression, which is associated with a reduction in Rb phosphorylation. FaDu and A253 cells were treated with the indicated concentrations of pevonedistat for 24 h. Phospho-Rb, total Rb, E2F1, and p21 levels were determined by immunoblotting. D E2F1 knockdown results in decreased DDB2 expression. E2F1 was silenced by lentiviral shRNA. The expression levels of E2F1 and DDB2 were measured by immunoblotting.

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: Inhibition, Quantitative RT-PCR, Expressing, Activity Assay, Transfection, Luciferase, Stable Transfection, Construct, Control, Phospho-proteomics, Western Blot, Knockdown, shRNA