Human telomerase reverse transcriptase (hTERT) promoter‐dependent conditionally replicative adenovirus (hTERT‐CRAd), which contains E1 genomes, allowed in‐trans replication of Ad‐NK4 DNA, leading to the co‐production of Ad‐NK4 particles. (A) Quantitative analysis of Ad‐NK4 DNA after co‐infection. SUIT‐2 cells were infected with Ad‐NK4 at 10 MOI and either Ad5/3hTERTLuc or Ad5/3hTERTE1 at 0.01 MOI simultaneously. Total DNA was extracted at the indicated time after co‐infection. Ad‐NK4 DNA was measured by qPCR and normalized by the corresponding levels of 18S DNA. Each value represents the mean ± SD of three independent samples. *P < 0.05, **P < 0.01. (B) Co‐produced Ad‐NK4 particles were released into the culture media and allowed the bystander effect. SUIT‐2 cells were infected with Ad‐NK4 (10 MOI) and either Ad5/3hTERTLuc (0.01 MOI or 0.1 MOI) or Ad5/3hTERTE1 (0.01 MOI or 0.1 MOI). After 48 h, each media was collected. Then, fresh SUIT‐2 cells were infected with each collected media, and after 48 h, the levels of NK4 expression in the new collected supernatants were measured using ELISA. Each value represents the mean ± SD of three independent samples. **P < 0.01.