The SHSY-5Y cell line (American Type Culture Collection) was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 300 mg/l L-glutamine, 4.5 mg/l D-glucose, 10 mg/l sodium pyruvate, 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution. Cells were seeded (2×10⁵ cells/ml) and maintained at 37°C in a 5% CO₂ humidified incubator. The culture medium was changed every 2 days. For subsequent experiments, cells were treated with various concentrations of ketamine (Fujian Gutian Pharmaceutical Industry Co., Ltd.) and incubated at 37°C for 12 and 24 h.

The effects of different concentrations of ketamine (0, 400, 800, 1,200 and 1,600 µM) on cell proliferation-neurotoxicity (viability) were assessed by performing the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). It has been reported that high doses of ketamine are required to induce SH-SY5Y cell apoptosis and the pre-experiment results indicated that 1,600 µM induced cell death but not excessive cell death (22). Cells were seeded (1×10⁴ cells/well) into 96-well plates and maintained at 37°C with 5% CO₂ for 24 h. Cells were treated with ketamine for 12 or 24 h at 37°C. Hydrogen peroxide (H₂O₂; 400 mM) was used as a positive toxicity control for 12 or 24 h at 37°C (23). Cell viability was assessed using the CCK-8 assay, according to the manufacturer's protocol. Briefly, 10 µl CCK-8 solution was added to each well and incubated at 37°C with 5% CO₂ for 1.5 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. At 48 h post-transfection, cells were selected using puromycin (1 mg/ml), this process took ~2 or 3 days, cells harvested after selection were used for subsequent experiments.

Lentiviral infection was used to increase the expression level of YAP in SH-SY5Y cells. LV5 (cat. no. A2831-3; Shanghai GenePharma Co., Ltd.) was used for YAP overexpression lentiviral particle production and LV5-negative control (NC) was used as the control for the YAP overexpression plasmid. LV5-based lentiviral vectors (1 µg/µl) were transfected into 293T cells at 80–90% confluence, 3.5×10⁵/ml. After purification and titration, the viral supernatant was harvested at 48–72 h post-transfection, passed through a 0.45-mm filter and diluted 2:3 with fresh medium containing 8 mg/ml polybrene. At 80% confluence, the SH-SY5Y target cells were infected with the viral supernatant. At 48 h post-transfection, cells were selected using puromycin (1 mg/ml). SH-SY5Y cells were seeded (0.5×10⁵ cells/well) into 24-well plates and incubated at 37°C for 24 h in a 5% CO₂ humidified incubator. Subsequently, the cell culture medium was aspirated and the virus was diluted in DMEM containing 10% FBS and 5 µg/ml polybrene. Blank and negative control (treated with the LV5NC plasmid) groups were established. After incubation for 12–24 h at 37°C, the viral suspension was removed, and DMEM was added before incubation at 37°C with 5% CO₂ for 24–48 h.

An siRNA was used to reduce the expression level of YAP in SH-SY5Y cells. The siRNAs (Shanghai GenePharma Co., Ltd.) used in the present study were as follows: YAP1-homo-1858 siRNA forward, CUGCCACCAAGCUAGAUAATT and reverse, UUAUCUAGCUUGGUGGCAGTT; and NC-siRNA forward, UUCUCCGAACGUGUCACGUTT and reverse, ACGUGACACGUUCGGAGAATT . The siRNA targeting YAP was fluorescently labeled and si-fluorescein amidite (FAM), which has an excitation wavelength of 480 nm and a emission wavelength of 520 nm, was used as the negative control. Cells were transfected with 20 µM YAP-siRNA or NC-siRNA using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C, according to the manufacturer's protocol. Briefly, 0.5–2×10⁵ cells and 500 µl DMEM without penicillin/streptomycin was added to each well and incubated for 24 h. Lipofectamine^® 2000 was diluted in 50 µl Opti-MEM I Reduced Serum medium (Gibco; Thermo Fisher Scientific, Inc.), mixed gently and incubated at room temperature for 5 min. Subsequently, 2 µl si-FAM diluted in 50 µl Opti-MEM I Reduced Serum medium was added to the Lipofectamine^® 2000 dilution and the cells incubated for 20 min at room temperature; YAP-siRNA prepared in the same way. The time interval between transfection and subsequent experimentation was 48 h.

After aspirating the culture media, cells were gently rinsed with PBS and fixed with 4% paraformaldehyde at 25°C for 15 min. Cells were rinsed twice with warm PBS and permeabilized using PBS containing 0.2% Triton X-100 and 0.2% BSA [Yeasen Biotechnology (Shanghai) Co., Ltd.] on ice for 15 min. Cells were blocked with PBS containing 0.02% Triton X-100 and 5% BSA for >30 min at room temperature. Subsequently, cells were incubated at 4°C overnight with an anti-cleaved caspase-3 (cat. no. 9661; 1:400; Cell Signaling Technology, Inc.) primary antibody. Following primary incubation, cells were incubated for 1 or 2 h at room temperature with Alexa 488-conjugated anti-rabbit IgG (cat. no. A11034; 1:300; Invitrogen; Thermo Fisher Scientific, Inc.) and 594-conjugated anti-rabbit IgG (cat. no. A21207; 1:300; Invitrogen; Thermo Fisher Scientific, Inc.) secondary antibodies, then cleaved caspase-3 protein labeled with the green fluorophore of the secondary antibody. Finally, nuclei were stained using DAPI (cat. no. 62248; 1:1,000; Thermo Fisher Scientific, Inc.) at room temperature for 10 min and sealed with 90% glycerin. Stained cells were visualized using a LSM88 confocal microscope (Zeiss GmbH) at magnifications of ×200, 400 and 630, ZEN 2.3 lite (Zeiss GmbH) was used for image capture and analysis.

SH-SY5Y cell apoptosis following ketamine treatment was analyzed by flow cytometry. Cells were seeded (3×10⁵ cells/ml) into 6-well plates and incubated at 37°C with 5% CO₂ for 24 h. Cells were treated with ketamine for 12 or 24 h at 37°C, harvested and washed twice with PBS. Subsequently, 5–10×10⁴ cells were re-suspended in 1X binding buffer. To each 5-ml culture tube, 100 µl cell suspension (1×10⁵ cells), 5 µl APC Annexin V and 5 µl 7-aminoactinomycin D (7-AAD) were added (cat. no. 561012, BD Pharmingen). Cells were incubated for 15 min at room temperature in the dark with gentle agitation. Subsequently, 400 µl 1X binding buffer was added to each tube and samples were analyzed by flow cytometry within 1 h. The following controls were used to set up compensation and quadrants: Unstained cells, cells stained with APC Annexin V and cells stained with 7-AAD, early and late apoptosis was assessed. FlowJo v10 (FlowJo LLC) was used for analysis and the model and supplier of the instrument used for flow cytometry was a Sony SA3800-Spectral Cell Analyzer (Sony Biotechnology).

Western blotting was performed as previously described (24). Briefly, total protein was extracted from ketamine-treated cells using RIPA buffer (Beyotime Institute of Biotechnology) containing phenylmethylsulfonyl fluoride, phosphatase inhibitors and protease inhibitors. Subsequently, the samples were centrifuged at 16,363 × g at 4°C for 10 min. Total protein was quantified using the bicinchoninic acid method. Protein (25 mg per lane) was separated via 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.) using the Trans-Blot Turbo Transfer system (Bio-Rad Laboratories, Inc.). After 90 min, the membranes were blocked with 5% BSA [Yeasen Biotechnology (Shanghai) Co., Ltd., cat. no. 36101ES76] in 1X Tris-buffered saline containing 0.1% Tween-20 (TBST; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Subsequently, the membranes were incubated at 4°C overnight with primary antibodies (diluted in 5% BSA in 1X TBST) targeted against: Cleaved caspase-3 (Asp175; cat. no. 9661; 1:1,000; Cell Signaling Technology, Inc.), Bax (cat. no. 2772; 1:1,000; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), YAP (cat. no. 4912; 1:1,000; Cell Signaling Technology, Inc.), β-tubulin (cat. no. 2146; 1:5,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. 4967; 1:5,000; Cell Signaling Technology, Inc.). Following primary incubation, the membranes were washed three times for 10 min with 1X TBST and incubated with the Goat anti-Rabbit IgG (H+L; cat. no. 31460; 1:5,000, Thermo Fisher Scientific, Inc.) secondary antibodies at room temperature for 1 h. The membranes were washed three times for 10 min with 1X TBST and protein bands were visualized by chemiluminescence (cat. no. RPN 2322, Cytiva) and analyzed with Image Lab v.5.2.1 62311 (Bio-Rad Laboratories, Inc.). β-tubulin and β-actin were used as the loading controls.

All experiments were repeated at least three times. Data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). Comparisons between two groups were analyzed using paired Student's t-test. Comparisons among multiple groups were analyzed using one-way or two-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The CCK-8 assay was used to investigate the optimum dose and exposure time of ketamine-induced cytotoxicity in SH-SY5Y cells. Different concentrations of ketamine induced cell toxicity, and cell viability was suppressed following treatment with ketamine for 12 and 24 h compared with the 0 µM group, particularly at a concentration of 1,600 µM. However, extensive cell death was observed when cells were treated with 2,000 µM ketamine (data not shown). Ketamine attenuated SH-SY5Y cell viability in a dose-dependent manner under the 24 h conditions. Short exposure to ketamine (12 h) also induced neurotoxicity in SH-SY5Y cells, but was less compared with 24 h. Nevertheless, cell viability was markedly decreased in a dose-dependent manner when the exposure time was extended to 24 h (P<0.01; Fig. 1A and B).

To further investigate whether ketamine-induced inhibition of cell viability was associated with apoptosis induction, western blotting assays were performed to measure the expression levels of apoptotic markers. SH-SY5Y cells treated with higher concentrations of ketamine displayed increased expression levels of cleaved caspase-3 and Bax, and decreased expression levels of Bcl-2 compared with the 0 µM group (Fig. 2A and B). Similar results were obtained for the immunofluorescence and flow cytometry assays. Cleaved caspase-3 protein expression levels (labeled with green fluorescent protein) and the rate of cell apoptosis increased with increasing ketamine concentrations (Fig. 2C-E). The results indicated that ketamine inhibited cell viability in a dose-dependent manner, which was associated with the activation of apoptosis.

To investigate the potential role of YAP in regulating the activity and viability of ketamine-treated SH-SY5Y cells, the present study induced YAP overexpression by lentiviral infection. The rate of apoptosis was decreased in the YAP overexpression group compared with the NC group (P<0.001; Fig. 3A and B). The CCK-8 assay indicated that YAP overexpression increased SH-SY5Y cell activity and viability following treatment with ketamine for 12 or 24 h compared with the NC group (P<0.05; Fig. 3C and D). Therefore, the results indicated that YAP overexpression protected SH-SY5Y cells against ketamine exposure.

To improve the current understanding of the role of YAP in ketamine-induced apoptosis, western blotting was performed to measure the expression of apoptotic markers, including cleaved caspase-3, Bcl-2 and Bax. Following YAP overexpression, SH-SY5Y cells were treated with 800 or 1,600 µM ketamine for 24 h. Following YAP overexpression, the expression levels of cleaved caspase-3 and Bax were decreased (P<0.01; Fig. 4A-D), whereas Bcl-2 expression levels were increased (P<0.05; Fig. 4C and D), compared with the NC group.

Flow cytometry was performed to assess ketamine-induced alterations to SH-SY5Y cell apoptosis. Compared with the NC group, ketamine-induced apoptosis was reduced following YAP overexpression. Furthermore, ketamine treatment increased SH-SY5Y cell apoptosis in a dose-dependent manner; however, YAP overexpression decreased the rate of ketamine-induced apoptosis at each concentration compared with the NC group (Fig. 5A). Similarly, the immunofluorescence results indicated that ketamine-induced cleaved caspase-3 expression was significantly decreased in the YAP overexpression group compared with the NC group (P<0.001; Fig. 5B and C). Therefore, the results suggested that YAP overexpression decreased ketamine-induced SH-SY5Y cell apoptosis.

To further investigate whether YAP knockdown altered ketamine-induced cell apoptosis, YAP expression was knocked down using an siRNA. Transfection efficiency of the siRNA was confirmed by performing western blotting (P<0.01; Fig. 6A and B). In addition, the expression levels of cleaved caspase-3, Bcl-2 and Bax in SH-SY5Y cells following treatment with ketamine for 24 h were measured. Cleaved caspase-3 and Bax expression levels were increased in the YAP knockdown group compared with the NC group (P<0.05; Fig. 6A, C and D). In addition, the expression levels of the anti-apoptotic protein Bcl-2 were decreased in the YAP knockdown group compared with the NC group (P<0.05; Fig. 6A and E). Therefore, the results suggested that YAP knockdown aggravated ketamine-induced apoptosis.