Codon optimization improves the bystander effect of the NTR/CB1954 system. (A) At 5% NTR-positive (ntr+) HEK-293 cells, only pGNTRo led to a detectable bystander effect, starting at a CB1954 concentration of 5 μM 48 hours after treatment. The effect of pGNTR became detectable at 25% ntr+ cells and 25 μM prodrug. (B) SHP-77 expressing gntro showed a significant bystander effect only at 25% ntr+ cells and 25 μM, while gntr-expressing cells did not. (A, B) The 100% ntr+ control experiments consistently reproduced the results obtained in Figure 5A and C. The data are normalized to the corresponding CB1954-untreated cell pools. Shown is one representative experiment out of two repetitions. *: p < 0.05; n = 3.