Fig. 4.

Lentiviral shRNA-mediated knockdown of c-Myc in endothelial cells causes down-regulation of Bmi-1 and up-regulation of p16. (A) Exponential phase AG10770 cells were infected with c-Myc shRNA-expressing or empty vectors, total RNA was extracted 5 days later, and c-Myc, Bmi-1, and p16 mRNA levels were quantified by qPCR. (B) Just-confluent AG10770 cells were infected with c-Myc shRNA-expressing or empty vectors and held under contact inhibition for 48 h; then the monolayers were scratch-wounded, incubation of cultures was continued for 24 h, and p16 protein was visualized by IHC staining. Cells were counterstained by using 0.1% crystal violet in 20% ethanol before image collection. (Upper) Intact monolayers. (Lower) Scratch wound. (C) Quantification of p16-positive cells in the intact monolayer (monolayer) and immediately adjacent to the denuded area (wound).