## 3 Results
### Apoptosis 6 h after IRE  
The efficiency of IRE was estimated by the induction of cell death or apoptosis 6 h after IRE. Cell viability, membrane integrity, mitochondrial morphology, and transcription of apoptosis-related genes were examined. Cell viability was reduced according to the applied electric field strength, and it reduced more significantly with pre-treatment from NIR (Figure 3a). We confirmed that this reduction is related to apoptosis. apoptotic cells were classified with positively stained Annexin-V FITC, and the apoptotic rate was significantly enhanced by pre-treatment with NIR (Figure 3b). TEM images showed that mitochondria in NIR+IRE cells were severely distorted, which implies accelerated apoptosis (Figure 3c). Mitochondrial degeneration occurs with the rupture of the mitochondrial outer membrane, as indicated by the white arrows. The rupture of the mitochondria was observed partially and entirely in HeLa cells that were under IRE and NIR-IRE. Entire ruptures were much more likely with NIR-IRE than with IRE.  
Figure 2: The 3 J/cm\({}^{2}\) NIR pre-treatment enhanced plasma membrane disintegration, right after IRE in HeLa cells. (**a**) Simulations of electric fields between two electrodes. (**b**) Relative MFI of Annexin-V dye according to the electric field intensity w/or w/o NIR pre-treatment. (**c**) Relative MFI of PI dye cells according to electric field intensity w/or w/o NIR pre-treatment. (**_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001 with the Student’s _t_-test to the control, # \(p\) \(<\) 0.05 and ## \(p\) \(<\) 0.01 with the Student’s _t_-test to the non-irradiated group (_n_ = 3)).  
Such apoptotic phenomena also appears at the gene level. We confirmed the increased transcription levels of apoptosis-related genes, BAX (Bcl-2-associated X protein), and p53 (Figure 3d). BAX is a pro-apoptotic protein that induces the opening of the mitochondrial membrane ion channel, and the release of cytochrome c. p53 is activated in response to many stress stimuli and directly regulates the transcription of apoptosis-related genes, including BAX [18]. HeLa cells have no mutations in p53 [19]. Additionally, we examined the transcription levels of oxidative stress-related genes: SOD1 (superoxide dismutase 1) and glutathione reductase (GSR). SOD1 is responsible for destroying excessive free  
Figure 3: The NIR pre-treatment enhanced apoptosis ratio after 6 h from IRE in Hela cells. 3 J/cm\({}^{2}\) NIR was irradiated, and then 1200, 1500, and 1800 V/cm electric pulses were applied. (**a**) WST-1 assay according to electric field intensity w/or w/o NIR pre-treatment. (**b**) Apoptosis rate according to electric field intensity w/or w/o NIR pre-treatment. (**c**) Representative TEM images of mitochondria in IRE-treated HeLa cells w/or w/o NIR pre-treatment. (**d**) Relative mRNA expression levels of BAX, p53, SOD1, and GSR. (**e**) WST-1 assay w/or w/o NAC. (* \(p\) \(<\) 0.05, **_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001 with the Student’s _t_-test to the control, ***_p_ \(<\) 0.01 with the Student’s _t_-test to the non-irradiated group, (_n_ = 3)).  
superoxide radicals by converting them to molecular oxygen and hydrogen peroxide in the outer mitochondrial membrane, and GSR is a key member of the glutathione antioxidant defense system. The reduction in the expression of these genes implies that the increase in apoptosis was not initiated by high oxidative stress in NIR pre-treated samples. This was confirmed by the addition of an antioxidant, which is widely used as a pharmacological antioxidant. The decline in cell viability by NIR+IRE was not recovered by treatment with NAC (Figure 3e). These results suggest that enhanced IRE-induced apoptosis was not due to enhanced oxidative stress.