RAd-delivered CD40L acts in synergy with conditionally replicating adenovirus. (A) RAdncCD40L cytotoxicity is dose-dependent. EJ cells were infected with 0, 10 or 100 MOI RAdncCD40L and 48 hours later cell viability was assessed by WST-1 assay. Results are the mean of triplet samples +/-SD. Two-tailed t-test 10 MOI vs. 100 MOI: p = 0.0001. Co-infection with RAdncCD40L and E1AΔCR2 enhances ncCD40L expression and cytotoxicity. (B) EJ cells were infected with 1 MOI RAdncCD40L and co-infected with 0, 0.1 or 1 MOI E1AΔCR2. Cell lysates were harvested at 48 and 120 hours and 20 μg total protein was blotted for ncCD40L. (C) EJ cells were plated in 96-well plates and infected with 1 MOI of either RAdncCD40L or RAdMock or left uninfected; simultaneously cells were infected with 0.1 MOI E1A ΔCR2 and 120 hours later cell viability was assessed by WST-1 assay. Results are the mean of triplet samples +/-SD. Two-tailed t-test E1A-CR2+RAdM vs. E1A-CR2+RAdncL: p = 0.0015.