Journal: EBioMedicine

Article Title: Ubiquinol-cytochrome C reductase core protein II promotes tumorigenesis by facilitating p53 degradation

doi: 10.1016/j.ebiom.2019.01.002

Figure Lengend Snippet: QCR2 is associated with p53 destabilization. (a) Western blotting for p53 and p21 in A549 cells transfected or infected with NC siRNA and Control Ad, NC siRNA and QCR2-Flag Ad, QCR2 siRNA-2 and Control Ad, or QCR2 siRNA-2 and QCR2-Flag Ad. Control Ad: adenovirus with an empty pcDNA vector control; QCR2-Flag Ad: adenovirus with a vector harboring QCR2-Flag. (b) Quantitative analysis of p53, p21, and QCR2 protein levels from the western blotting results shown in (a) using ImageJ. (c) Western blotting for indicated proteins in the hepatic cell line QSG7701 cells treated as described in (a). (d) Western blotting for p53, p21, and QCR2 in A549 cells transfected with NC siRNA, NC siRNA and QCR2 siRNA-2, QCR2 siRNA-2 and p53 siRNA, or NC siRNA and p53 siRNA. (e) RT-qPCR analysis for CDKN1A mRNA in cells treated as described in (c) (*** p < .001, student's t -test). (f, g) A549 cells were transfected with NC siRNA or QCR2 siRNA-2 (f) and Control Ad or QCR2-Flag Ad (g) in the presence or absence of a proteasome inhibitor, PS-341. The transfected cells were analyzed by western blotting for p53 and QCR2. (h, i) A549 cells were transfected with NC siRNA or QCR2 siRNA-2 (h) and Control Ad or QCR2-Flag Ad (i). The cell lysates were immunoprecipitated with an anti-p53 antibody, and immunocomplexes were analyzed by western blotting for p53 and ubiquitin (Ub). (j) A549 cells were treated with 1 μg/μL of a protein synthesis inhibitor, cycloheximide (CHX), at the indicated time points after transfection with QCR2 siRNA-2 or NC siRNA for 72 h. Proteins were extracted and subjected to western blotting for p53, QCR2, and GAPDH.

Article Snippet: For overexpression of QCR2, a recombinant adenovirus vector expressing QCR2 (GenBank accession number NM_003366 ) or empty pcDNA control was provided by Vigene Biosciences (China).

Techniques: Western Blot, Transfection, Infection, Plasmid Preparation, Quantitative RT-PCR, Immunoprecipitation