Fig. 2: Kinetics and reversibility of iCROP-modified fLuc delivered as DNA or mRNA.

a Schematic representation of the post-translational steps that iCROP-fLuc undergoes in the presence or absence of the protease inhibitor. b FLuc luminescence every 15 to 30 min during 90 min of incubation with rupintrivir at the indicated concentrations. HEK293T cells were transfected with iCROP-fLuc (P_mPGK-HRVp_ΔQ182-fLuc_CS-K491-pA) and 36 h later the medium was exchanged for fresh medium either without or with rupintrivir. c Real-time fLuc analysis from HEK293T cells expressing iCROP-fLuc. The recording of the signal from live cells was started 90 s before changing the medium to fresh medium either without or with rupintrivir (100 nM or 1000 nM). d iCROP-fLuc reversibility. HEK293T cells expressing iCROP-fLuc were alternated between rupintrivir-containing (100 nM) and rupintrivir-free media. Each rupintrivir induction lasted 45 min, followed by 135 min in rupintrivir-free medium. fLuc intensity was measured at the indicated time points. e Inducibility of iCROP-fLuc delivered as mRNA. Luminescence from HEK293T cells transfected with in vitro-transcribed mRNA coding for iCROP-fLuc for 6 h, followed by incubation for additional 6 h in the presence of the indicated rupintrivir concentrations. f Activation kinetics of mRNA-delivered iCROP-fLuc. HEK293T cells simultaneously transfected with iCROP-fLuc mRNA and treated with rupintrivir (1 μM) were analyzed for fLuc intensity at the indicated time points. In b–f, data are shown as mean ± SD (n = 3 biological replicates). Statistical significance was calculated by means of Welch’s two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.