Fig. 1: Design of the direct-current-activated transgene expression switch in mammalian cells.

a, Schematic illustration of the stimulation setup for monolayer cultures. Each well of a 24-well plate has two platinum wires that function as anode and cathode, placed 0.6 cm apart submerged in the culture medium. When electric current is applied, bubbles form around the electrodes, with production of chlorine gas at the anode and hydrogen gas at the cathode. b, Schematic representation of the electrogenetic circuit based on the NRF2/KEAP1 antioxidative response. Upon electrical stimulation, the formation of ROS is sensed by constitutively expressed NRF2 and KEAP1 complexes localized in the cytoplasm, which triggers the translocation of NRF2 to the nucleus, where it activates expression of the gene of interest by binding to ARE sites in the upstream synthetic promoter. Under non-stimulating conditions, NRF2 is continuously targeted to the 26S proteasome for degradation. c, SEAP produced by transiently transfected HEK293 cells (KEAP1, pJH1004; NRF2, pJH1003; P_DART-SEAP, pJH1005) upon stimulation by DC with 10 V for 15 s (DC10V) and 5 V for 20 s (DC5V). d, SEAP produced by cells transfected with only ARE reporter (P_DART-SEAP, pJH1005) or together with KEAP1 (pJH1004) and NRF2 variants (wild-type NRF2, pJH1003; NRF2-VP64, pJH1175) and reporter (pJH1005). Cells were stimulated with DC5V for 20 s. e, SEAP produced by cells co-transfected with KEAP1 (pJH1004), NRF2 fused to tetracycline-dependent transactivator TetR-VP64 (NRF2-TetR-VP64, pJH1181) and the cognate reporter (P_TRE-SEAP-pA, pMF111). The cells were stimulated with DC5V for 20 s. f, SEAP produced by cells co-transfected with reporter constructs containing one (DART1), two (DART2), three (DART3) and four (DART4) ARE repeats in the promoter region and stimulated with DC5V for 20 s. Data are mean ± s.d., n = 4. P values were calculated between stimulated and non-stimulated controls.

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