Depletion of Human Api5 in p16^INK4a-Deficient Squamous Cell Carcinoma Cells Results in Reduced Survival versus Normal Human Fibroblasts

<div><p>(A) RNAi-mediated depletion of transfected Api5. Various shRNA constructs were tested for their ability to deplete FLAG-Api5 transfected U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfected constructs strongly depleted Api5 expression after 3 d as determined by anti-FLAG immunoblot.</p><p>(B) RNAi-mediated depletion of endogenous Api5 protein. JHU-029 cells were infected with lentiviral encoding scramble or API5-AB shRNAs and selected with puromycin for 3 d. Expression of endogenous Api5 was determined by immunoblotting with affinity-purified anti-Api5 polyclonal antibody (G3162).</p><p>(C and D) Endogenous Api5 expression in JHU-029 cells is nuclear and excluded from the nucleolus. JHU-029 cells were stained with the G3162 polyclonal antibody after 4% paraformaldehyde fixation.</p><p>(E) Api5 RNAi reduces survival of JHU-029 tumor cells as compared to normal human fibroblasts. JHU-029 cells, as well as normal human WI38 diploid fibroblasts, were infected with lentiviral empty vector or vectors encoding API5-AB shRNAs for 24 h and plated onto culture plates in 10% fetal calf serum–containing media. Cell survival was determined by MTT assay at the indicated days post–lentiviral infection. Api5 and control actin expression was determined from equally loaded protein from day 3 lysates.</p>