The TE354T BCC and HaCaT keratinocyte cell lines were acquired from The American Type Culture Collection, and were maintained in DMEM/High Glucose (HyClone; GE Healthcare Life Sciences; pH 7.0-7.4; buffer salt including sodium phosphate monobasic, sodium bicarbonate and HEPES) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.; 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured at 37˚C and 5% CO₂ in a humidified environment.

CAP equipment was provided by The Institute of Plasma Physics, Chinese Academy of Sciences (Fig. 1). The air plasma device includes a DC voltage input, a copper rod array and a ballast resistor. The electrode consists of six arrays of thin copper rods with a radius of 1 mm. The ballast resistors are 30 Ω, and the discharge current is limited to ~5 mA. The device is activated by a 10-kV DC voltage and generates plasma through a copper rod electrode. DMEM or PBS was added to a 35-mm cell culture dish and irradiated with plasma to prepare the PAS. Each dish was filled with 2 ml liquid (DMEM or PBS), the plasma electrode was placed ~10 mm from the liquid surface, and plasma activation liquids of different concentrations were prepared by irradiation for different times (60, 120, 180, 240 and 300 sec). The doses of plasma-activated liquid are defined as the different processing times of the liquid under the same discharge parameters. The prepared activated solution was placed in an ice water bath or stored at 4˚C for further experiments.

TE354T cells were seeded in 6-well plates (2x10⁵ cells per well) and incubated at constant temperature for 24 h. The medium was aspirated and 2 ml of 180 sec plasma-activated DMEM solution was added to each well for incubation for 6 and 12 h. The morphological changes of the cells were observed directly under a light microscope (magnification, x400). A Hoechst 33342 Staining kit was purchased from Beyotime Institute of Biotechnology. The sterilized coverslips were placed in 6-well plates and inoculated with TE354T cell culture. After the cells had grown to 50-80% of the coverslip area, the medium was aspirated and the cells were treated with 2 ml 180 sec plasma-activated DMEM solution for 2 h. DMEM was aspirated again and the fixing solution (cat. no. C0003-1; Beyotime Insitute of Biotechnology) was added at 4˚C for 10 min. Subsequently, the cells were rinsed twice with PBS and 0.5 ml Hoechst 33342 staining solution was added for incubation at room temperature for 5 min in the dark. The cells were washed twice with PBS and imaged under a fluorescent microscope for observation (magnification, x1,000). The images were taken using an IX-71 Olympus microscope (Olympus Corporation).

Cell viability was measured by an MTT assay. All experiments used logarithmic growth phase cells collected by digestion, which were counted and inoculated in a 96-well culture plate for 24 h (10,000 cells; 100 µl culture medium per well). The cells were fully adherent at the start of experimental treatment. The cells were stimulated with plasma-activated fluid for 2 h. Thereafter, the activation fluid was replaced with fresh DMEM and incubation was continued for 24, 48 or 72 h. In the 96-well plates, 100 µl MTT solution was added to each well for 4 h. Thereafter, the formazan was dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader (SpectraMax i3x; Molecular Devices, LLC).

An ROS assay kit was purchased from Beyotime Institute of Biotechnology. HaCat and TE354T cells in the logarithmic growth phase were inoculated on 96-well culture plates (10,000 cells per well), and the experiment was performed after the cells were completely adherent. Cells were loaded with the fluorescent probe dichloro-dihydro-fluorescein diacetate and stimulated with plasma-activated DMEM or PBS, and changes in the concentration of ROS were measured after 2 h. Fluorescence was measured using a fluorescence plate reader (SpectraMax i3x; Molecular Devices) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm to calculate intracellular ROS levels.

A H₂O₂ assay kit was purchased from Beyotime Institute of Biotechnology. The cell-free DMEM and PBS solutions were placed in 2-ml culture dishes, and were exposed to CAP treatment for 60, 120, 180, 240 and 300 sec, respectively. H₂O₂ detection reagent was added and a microplate reader (SpectraMax i3x; Molecular Devices) was used to measure A560 to calculate the H₂O₂ concentration in the liquid. The pH changes in different experimental groups were detected using a pH meter. NO₃⁻ in the PBS after plasma treatment was evaluated using PhotoLab 6100 (Merck KGaA) with nitrate detection kit (Merck KGaA).

The Annexin V-FITC/propidium iodide (PI) apoptosis kit was purchased from Best-Bio(http://bestbio.bioon.com.cn/), and a flow cytometer was provided by The Institute of Technology Biology, Chinese Academy of Sciences. Cells were innoculated in 6-well plates and were cultured until fully adherent, and then stimulated for 2 h in PAS; thereafter, the medium was replaced with fresh medium and samples were placed in the incubator for 24 h. The cells were collected for staining. Cells were suspended in Annexin V-FITC binding solution at a concentration of ~1x10⁶/ml. Subsequently, 5 µl Annexin V-FITC staining solution was added for incubation for 15 min. A total of 10 µl PI staining solution was then added for incubation for 5 min, protected from light at 2-8˚C. The apoptosis rate of each group of cells was analyzed using a flow cytometer (BD Biosciences). The data were analyzed using FlowJo software (v10.0.7; FlowJo LLC).

Western blot analysis was performed at 0, 3, 6 and 12 h after treating the cells with PAS. The cells were harvested by trypsinization and the resulting cell suspension was washed twice with PBS. RIPA (Beyotime Institute of Biotechnology) lysis buffer was added, and the supernatant was collected after centrifugation (4˚C; 10,000 x g; 5 min). Protein quantification was performed using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Loading buffer was mixed with protein and the mixture was boiled for 3 min. Subsequently, proteins (10 µl per lane) were separated by SDS-PAGE on 10% gels for 90 min, and transferred to a polyvinylidene fluoride film (Bio-Rad Laboratories, Inc.) at a temperature of 4˚C. The membrane was blocked with 5% skim milk powder in TBS with Tween-20 (TBST) for 1 h at 24˚C. Antibodies against Cas3 (cat. no. 9665T; Cell Signaling Technology, Inc.; 1:1,000), cleaved Cas3 (cat. no. 9664T; Cell Signaling Technology, Inc.; 1:1,000), Cas9 (cat. no. 9508T; Cell Signaling Technology, Inc.; 1:1,000), cleaved Cas9 (cat. no. 7237T; Cell Signaling Technology, Inc.; 1:1,000) and β-actin (cat. no. TA-09; OriGene Technologies, Inc.; 1:1,000) were added and incubated overnight at 4˚C. The films were then washed three times with TBST, and secondary antibodies conjugated with horseradish peroxidase (1:10,000) were added for incubation for 1 h at room temperature. The secondary antibodies used for Cas3, cleaved Cas3 and cleaved Cas9 were goat rabbit anti-lgG (cat. no. ZB2301; OriGene Technologies, Inc.); the secondary antibodies used for Cas9 were goat mouse anti-IgG (cat. no. ZB2305; OriGene Technologies, Inc.). Signals were detected with using an enhanced chemiluminescence kit (Amersham ECL plus; GE Healthcare Life Sciences). β-actin (was used as an internal control. Density was analyzed using the Fino-do X6 analysis system (Tanon Science and Technology, Co., Ltd.). ImageJ software (v1.46; National Institutes of Health) was used to quantify the protein bands of interest.

Cells were treated with PAS for 0, 4 or 8 h, followed by total RNA extraction using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cell lysate was loaded into an RNase-free centrifuge tube and RNA-seq was performed by Shenzhen Huada Gene Technology Co., Ltd. The sequencing results and analysis of differential gene expression functions were performed by Shenzhen Huada Gene Technology Co., Ltd. The sequencing results were provided by Shenzhen Huada Gene Technology Co., Ltd. The analysis results report has not been shared to a public database at the time of publication.

Data are presented as the mean ± SD of three independent experiments. Statistical significance was determined using an independent Student's t-test and ANOVA. Least Significant Difference post hoc test and Dunnett's post hoc test were used where appropriate. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism (version 5.01; GraphPad Software, Inc.) software.

Compared with untreated cells, cells treated with PAS for 6 h exhibited reduced intracellular volume and shrunken cell membranes. Furthermore, certain cells had detached from the bottom of the culture dish, and appeared small and round (Fig. 2). These effects became more apparent after treatment for 12 h. Hoechst staining revealed bright blue cells after PAS treatment, indicating that the nucleus was condensed, and that the cells had begun to undergo apoptosis.

Increased PAS treatment time resulted in a gradual decrease in the cell viability rate and this effect was more pronounced in the PBS group compared with the DMEM group (Fig. 3). Furthermore, plasma-activated DMEM and PBS stored for 24 h prior to treatment induced cell death. It is worth noting that after PAS storage for 24 h, compared with the control group, the CAP-treated DMEM and PBS for 60 sec showed a proliferation-promoting effect on the cells.

As presented in Fig. 4, following treatment with PAS, TE354T cells were cultured for 24, 48 and 72 h under standard conditions. The survival rate of each group was then measured. It was observed that the proliferation of TE354T cells was inhibited within 72 h of PAS treatment. In the 300 sec group, after TE354T cells were treated with CAP-activated DMEM and CAP-activated PBS, the cell survival rate was ~30%, and this inhibition continued to be significant for 72 h. Furthermore, PAS demonstrated dose- and time-dependent inhibitory effects on the proliferation of TE354T cells.

PAS significantly decreased the proliferation of TE354T cells compared with HaCat cells (Fig. 5). In the absence of malignant epidermal keratinocytes, the viability of cells within the same groups was significantly higher than those of the BCC cell groups. In the DMEM group, even at the highest dose of PAS treatment, the survival rate of HaCat cells was ~75%, while that of TE354T cells in the same group was <40%. In the PBS group, this difference was less pronounced. Compared with DMEM, the apoptotic effect of PBS following plasma irradiation was stronger, but the selectivity was relatively weak.

The intracellular ROS levels in HaCat and TE354T cells were examined before and after PAS treatment (Fig. 6). After treating cells with CAP-activated DMEM, ROS levels increased by ~1.7-fold in TE354T cells in the 300 sec group, while those in HaCat cells only increased by ~1.4-fold. After treating cells with CAP-irradiated PBS, ROS levels in TE354T cells were increased by a maximum of ~2.6-fold. The basal ROS concentration in HaCat cells was lower than that in TE354T cells, and the increase in ROS levels in HaCat cells was lower after PAS treatment.

After 60, 120, 180, 240 and 300 sec of CAP treatment, the pH of both DMEM and PBS solutions demonstrated a downward trend (Fig. 7A). After CAP treatment, H₂O₂ in both solutions increased. This growth trend was more pronounced in PBS solution after receiving CAP treatment at the same time. After 300 sec of CAP treatment, the H₂O₂ concentration measured in PBS solution was ~33 mg/l. At the same time, it was ~28 mg/l in DMEM (Fig. 7B). As the color of DMEM will affect the detection of NO₃-, only the change of NO₃- concentration in PBS solution after CAP treatment was measured. No NO₃- concentration was measured in the blank control group With the increase of CAP treatment time, the concentration of NO₃- in PBS solution increased (Fig. 7C). After 300 sec of treatment, the measured NO_3- concentration in the solution was ~90 mg/l.

Cell staining with Annexin V-FITC/PI can indicate the rate and time of apop-tosis in cells. In the flow cytometry plots, the lower and upper right quadrants indicate early and late apoptosis, respectively. As presented in Fig. 8, the apoptotic rate was significantly increased following PAS treatment compared with the untreated control groups. Both early and late apoptosis exhibited an upward trend, which was more pronounced in the PBS-treated group.

Cleaved-caspase (cle-cas) is the activated form of caspase (cas). The relative activity of cas9 and cas3 was significantly increased following PAS treatment, suggesting that PAS induced the intrinsic apoptotic pathway in TE354T cells. The ratio of cle-cas to cas was calculated, and the ratio increased significantly following PAS treatment. After 12 h, the activity of cle-cas9 demonstrated a downward trend, possibly due to the necrosis of cells (Fig. 9).

Differentially expressed genes (DEGs) were analyzed in untreated control cells and BCC cells stimulated with PAS for 4 or 8 h. An increase in the expression of 754 genes, and a decrease in the expression of 281 genes, were observed after stimulation with PAS for 4 h. After 8 h, the number of upregulated DEGs increased to 1,763 (Fig. 10A). Based on these results, the genes were classified according to Gene Ontology (GO) terms: Molecular function, cellular component and biological process (Fig. 10B). Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway classification and enrichment analysis was performed (Fig. 10C). Based on the degree of enrichment and the number of DEGs, 'MAPK signaling pathway', 'TNF signaling pathway' and 'IL-17 signaling pathway' were the KEGG pathways most signifi-cantly associated with PAS-induced apoptosis.