## 4 Discussion  
We successfully demonstrated that NIR pre-treatment enhanced IRE-induced apoptosis in vitro. The cell membrane was easily disturbed by electric pulses, which was shown by increased PS translocation and PI transportation with NIR irradiation (Figure 2). The apoptosis signal was enhanced, which was shown by decreased DNA synthesis, increased PS translocation, mitochondrial condensation, and increased apoptosis-related mRNA synthesis after 6 h from IRE (Figure 3a-d). In this study, we used 810 nm NIR light, which has demonstrated mitochondrial activation or intracellular ROS enhancement in previous reports [14,15]. However, our results showed that cellular responses to 810 nm NIR were dependent on the irradiation intensity. The CMP was hyperpolarized at 2-4 J/cm\({}^{2}\), and the MMP was hyperpolarized  
Figure 4: Capacitance and conductivity changes of the cells with NIR irradiation. (**a**) Schematics of impedance measurement of cell-cultured ITO glass device. (**b**) Relative capacitance changes with irradiation time. (**c**) Relative capacitance changes with irradiation time. at a lower intensity of 1-2 J/cm\({}^{2}\) (Figure 1c,d). Figure 1e shows a significant reduction in intracellular ROS at 3-5 J/cm\({}^{2}\). We used 3 J/cm\({}^{2}\) NIR irradiation for the experiments with IRE, which had no harmful effects on proliferation, induced CMP hyperpolarization, induced no effects on MMP, and reduced the ROS levels. Under these conditions, we hypothesized that NIR pre-stimulation promotes IRE-induced cell death through membrane hyperpolarization, rather than through ROS generation. Irrelevance of ROS in the enhanced apoptosis was confirmed with reduced antioxidant gene expression and with no recovery from antioxidant chemicals (Figure 3d,e). Therefore, we hypothesize that CMP hyperpolarization may be one of the reasons for the higher IRE-induced apoptosis. As the induced transmembrane potential caused by externally applied electric pulses is superimposed on the resting CMP of the cell, the side of the cell facing the anode is hyperpolarized, while the side facing the cathode is depolarized [21]. Previous studies reported that hyperpolarization yielded a more statistically significant electroporation efficiency than depolarization [14]. The addition of cationic peptides makes the anode-facing cell membrane more negative, and cations interact electrostatically with the plasma membrane, producing a greater negative charge, thereby increasing the electrostatic potential across the membrane. [PERSON] et al. showed that CMP can affect the threshold voltage that triggers electroporation [22]. [PERSON] et al. showed that CMP is positively correlated with electroporation-induced molecular transfer through the membrane [23; 24]. Although photobiomodulation is widely used in research and clinical fields, the mechanism by which NIR modulates cellular behavior is not yet clearly understood. Cytochrome c oxidase in the mitochondrial respiratory chain has been considered the primary photo-acceptor for photobiomodulation [14; 25; 26]. However, cellular complex responses cannot be explained simply by one chromophore [27; 28], and other potential mechanisms (e.g., interfacial water layer) and chromophores (e.g., light-sensitive ion channels) have been suggested [29; 30]. [PERSON] et al. suggested that pulsed NIR increased membrane capacitance and capacitive currents through temperature changes in a short time [13]. Our impedance measurements showed a slight decrease in capacitance and a slight increase in conductance with NIR irradiation. This indicates a change in the plasma membrane structure or an increase in charge. We speculate that this is another cause for the enhanced IRE efficiency, as well as the increased CMP. This phenomenon may include biological responses rather than physical responses. Our results also show that there are windows for hyperpolarization in the cytoplasmic and mitochondrial membranes and the latter seems more vulnerable to NIR stimulation.