Fig. 2. RELEASE in circuits.

a Orthogonal operation of RELEASE constructs. b HEK293 cells were co-transfected with SEAP fused to RELEASE (responsive to TEVP) and GFP fused to RELEASE (responsive to HCVP). SEAP and GFP levels increase in the supernatant when the cognate protease was expressed. c Tandem insertion of two protease cut sites (top panel) created a RELEASE construct that implemented OR gate logic. If either of the respective proteases were expressed, the dilysine ER retention motif would be removed, and SEAP would be secreted. d Implementation of AND logic by adding the N-terminal p450 signal anchor sequence as a second ER retention domain, so that both proteases would have to be present to remove both retention domains and allow SEAP to be secreted. e A two-protease cascade was created where TEVP was required to activate TVMVP (processing protease), which subsequently cleaved SEAP RELEASE. SEAP secretion increased when TEVP was expressed (right panel). Each dot represents an individual biological replicate. Mean values were calculated from three (b) or four replicates (c–e). Error bars represent ±SEM. The results are representative of at least two independent experiments; significance was tested by one-way ANOVA with a Tukey’s post-hoc comparison test among the multiple conditions. *p < 0.05, ****p < 0.0001.