PMC

NTS activates MMP-2/MMP-9. (A,B) NTS increases mRNA expression of MMP-2/MMP-9. NTS was generated by NTP treatment for 10 and 30 seconds. MMP-2/MMP-9 mRNA level was measured using real-time PCR. The mRNA expression of MMP-2/MMP-9 was significantly increased in the group treated with NTP for 30 seconds. Asterisks indicate statistically significant differences (*P < 0.05; ***P < 0.001). (C) NTS increases MMP-2/MMP-9 activity in bronchial epithelial cells. Gelatin zymography for MMP-2/MMP-9. Only MMP-2/MMP-9 of active form was statistically increased after NTS treatment. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.001).

NTS reduces the expression of E-cadherin, while the expression of slug increases. (A) NTS induces down-regulation of E-cadherin and up-regulation of Slug. NTS was generated by NTP treatment for 10 and 30 seconds. The degree of expression of E-cadherin and slug was confirmed by Western blot. The grouping of blots cropped from different parts of the same gel. (B,C) Relative intensity was identified and confirmed again. Relative intensity of E-cadherin decreased with NTP treatment time, while slug increased. Both values were statistically significant. Asterisks indicate statistically significant differences (**P < 0.01; ***P < 0.001).

NTS does not induce cytotoxicity in the bronchial epithelial cells and proliferation is increased. (A) NTS did not affect bronchial epithelial cells viability, but rather increased in proportion to the treatment time. NTS was generated by NTP treatment for 10 and 30 seconds in culture media, and was measured after culturing for 24 hours. Cell viability was evaluated by the MTT assay. (B) NTS increased bronchial epithelial cell proliferation. Statistically significant proliferation was increased with treatment time. Cell proliferation was measured with a BrdU assay. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.001).

NTS increases EGFR activity, not amounts of EGFR protein. (A) NTS increases the expression of p-EGFR. The expression level of p-EGFR and total EGFR was evaluated using the Western blot. NTS was generated by NTP treatment for 10 and 30 seconds. After NTS treatment, the expression level of p-EGFR was increased with NTP treatment time and the total EGFR expression was not changed. The grouping of blots cropped from different parts of the same gel. (B) NTS increases the relative intensity of p-EGFR and statistically increased with the treatment time. Asterisks indicate statistically significant differences (**P < 0.01; ***P < 0.001). (C) NTS does not affect changes in total EGFR. The total amount of EGFR protein after NTS treatment did not change statistically.

NTS increases the expression of proteins involved in EMT signaling. (A) Expression of proteins involved in EMT signaling was confirmed by Western blot. NTS was generated by NTP treatment for 10 and 30 seconds. The degree of protein expression of p-FAK and p-Src was significantly increased with NTP treatment time. The grouping of blots cropped from different parts of the same gel. (B) The increase in the expression of p-FAK after NTS treatment was confirmed using immunefluorescence assay. The red-stained stock in cytoplasm represents p-FAK (white arrow). The amount of red- stained stock was increased in the NTS treated group. (C) Fluorescence intensity of p-FAK statistically significantly increased by NTS. Asterisks indicate statistically significant differences (**P < 0.01).

NTS increases cell migration. (A) NTS increases cell migration. The cells were treated with NTS generated by NTP for 30 seconds and incubated for 24 hours. BEAS-2B cells were plated in 6-well plates and a scratch wound healing assay was performed. (B) NTS decreased the denuded zone of scratched area significantly. Mean denuded zone was obtained by calculating the ratio of average area of the denuded zone to the area in the control. Asterisks indicate statistically significant differences (***P < 0.001). (C) Transwell migration assay was performed after NTS treatment under the same conditions. (D) The number of migrated cells increased statistically. Cells were photographed using a fluorescence microscope and examined at ×10 magnification. Asterisks indicate statistically significant differences (***P < 0.001).

NTS improves the regeneration of nasal mucosal epithelium in animal models of nasal wound. (A) H&E staining of septal mucosa after nasal wound formation using nasal brushing technique. The basement membrane was well maintained and the wound was properly formed (black arrow). (B) H&E staining of septal mucosa after normal saline (control) and NTS treatment. NTS was generated by NTP treatment for 30 seconds. The thickness of the epithelial layer was increased in the NTS group. However, the thickness of the subepithelial layer was reduced. (C) ETI increased post-NTS treatment. Compared with the control group, the NTS treated group showed a statistically significant increase in ETI. (D) After NTS treatment, STI increased. Starting with day 5 of NTS treatment, STI was significantly decreased in NTS treated group. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.001). (E) Immuno-histochemical staining of septal mucosa after normal saline and NTS treatment. Higher expression of p-EGFR was observed in the NTS group.