Overexpression of CD86FLm provides a survival advantage against silencing of CD86 and CD28 but not against silencing of IRF4. (A) Representative histograms showing the levels of surface CD86 in 3 different cell lines when either CD28, CD86, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are silenced. Thin black histograms at left are unstained pLKO.1-infected controls. (B) Cell death measured at day 4 postinfection via annexin V staining, shown as percentage of pLKO.1-infected controls. (A-B) Data shown are from day 4 postinfection and representative of at least 3 independent experiments. (C) qRT-PCR was performed to determine levels of CD86, CD28, and IRF4 to compare the different cell lines. Data are normalized to β-actin as endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. Data shown are mean ± SEM of at least 3 independent experiments. (D) Representative western blots showing levels of IRF4 in cells overexpressing CD86FLm or CD86TLm when either CD86 or CD28 are silenced. Lysates are from day 4 postinfection. (E) Cell death as measured by annexin V staining at day 4 postinfection in 8226-pCDNA3.1 or 8226-CD86FLm cells where CD86 or IRF4 was silenced; shown as percentage of pLKO.1-infected controls. (F) Representative western blots showing levels of IRF4 and β-actin in lysates from experiments in panel E. Data shown are mean ± SEM of at least 3 independent experiments. *P < .05, **P < .01, ***P < .005.