STC-1 cells were obtained from the Guangzhou Jennio Biotech Co., Ltd. (Guangzhou China). Cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 2.5% (vol/vol) fetal bovine serum (Gibco, Carlsbad, CA, USA), 10% (vol/vol) horse serum (Gibco), 100 U/ml penicillin (Life Technologies, Carlsbad, CA, USA) and 100 U/ml streptomycin (Life Technologies), and incubated in a humidified atmosphere containing 5% CO₂ at 37°C. The medium was changed every 2–3 days. When the cells reached 80–90% confluence, they were trypsinized with 0.25% trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA) and subsequently replated into culture flasks. Each experiment was repeated at least three times (n≥3).

For the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay, 2×10⁴ cells/well were seeded in 96-well plates for 24 h at 37°C and starved for a further 24 h in serum-free DMEM. Subsequently, the cells were exposed to LPS (Escherichia coli 0111:B4; Sigma-Aldrich) at various concentrations (0, 0.1, 0.5, 1 and 10 μg/ml) at 100 μl/well. Following incubation for 24 h, 20 μl/well of MTT (Sigma-Aldrich) solution (5 mg/ml in PBS) was added and the cells were cultured at 37°C for 4 h. Subsequently, the supernatant was discarded carefully. The formazan crystals were dissolved in 100 μl/well of DMSO. The plates were agitated for 5 min, and the absorbance of solubilized formazan was measured at 570 nm using an automatic microplate reader (Multiskan MK3, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell survival was expressed as the ratio of the absorbance of treated cells to the untreated cells.

STC-1 cells (1×10⁶ cells/well) were exposed to various concentrations of LPS for 24 h. The cells were washed in PBS and stained with Hoechst 33258 (5 μg/ml) for 5 min at 37°C. The stained cells were washed twice in PBS and then observed immediately using a fluorescence microscope (Leica; Wetzlar, Germany). An Ex/Em filter set of BP330–380/LP420 nm was used, and the images were recorded using a color charge-coupled device camera.

Cells (2×10⁶) were seeded into six-well plates for 24 h and starved for a further 12 h in serum-free DMEM. Apoptotic cells were identified based on the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, measured using an annexin V/propidium iodide (PI) double staining apoptosis detection kit (Life Technologies). Following treatment with or without various concentrations of LPS for 24 or 6 h, the cells were washed three times with PBS and immediately fixed in 4% paraformaldehyde for 15 min or harvested and washed three times in cold PBS and resuspended in an annexin-binding buffer. Subsequently, the fixed cells were added with annexin V-fluorescein isothiocyanate (FITC) and PI buffer, while the resuspended cells were added to annexin V-FITC and PI directly. The reaction was incubated at room temperature for 15 min in the dark. Stained cells were analyzed immediately using an Accuri C6 flow cytometer (BD Biosciences; San Jose, California, USA) and a fluorescence microscope (Leica). Fluorescence was detected with an excitation wavelength of 488 nm.

For flow cytometry, the LPS exposure time was reduced to 6 h. This change was due to the fact that flow cytometry using annexin V staining is a more sensitive technique for detecting apoptosis compared with the other techniques used in our study.

In order to examine Bcl-2, Bax and caspase-3 protein levels, cells were grown in culture flasks and treated with LPS as described in a previous section. After washing in ice-cold PBS, cells were lysed in a lysis buffer [50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM phenylmethanesulfonylfluoride (PMSF); 1 mM EDTA; 1% Triton X-100; 1% sodium deoxycholate; and 0.1% sodium dodecyl sulfate (SDS); Sigma-Aldrich] at 4°C for 30 min. Cell lysates were centrifuged for 10 min at 15,400 rpm at 4°C, and the supernatants were collected. The protein concentration of the samples was estimated according to the BCA method (10). Subsequently, 50 μg of total protein from each sample was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. The membranes were blocked with 2% non-fat dry milk in PBS containing 0.25% Tween-20 overnight and incubated with Bcl-2, Bax (Cell Signaling Technology, Beverly, MA, USA) and caspase-3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) antibodies for 2 h at room temperature. Following the three washing steps, anti-horseradish peroxidase-conjugated secondary antibodies (Beyotime, Haimen, China) were applied, and the membranes were stained using enhanced chemiluminescence reagents (Beyotime). The autoradiographs were scanned and semiquantitatively quantified.

Data were presented as the means ± SD. Significant differences between groups were evaluated using one-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.

STC-1 cells were cultured in the absence and presence of varying concentrations of LPS for 24 h. The effects of LPS on cell viability were assessed by MTT assay (Fig. 1). Exposure to increasing concentrations of LPS induced a dose-dependent decrease in cell survival. At the highest tested concentration of LPS (10 μg/ml), the survival rate was 52±3% when compared with the control group. At 1 μg/ml LPS, cell viability was reduced to 64±12%. However, no significant growth inhibition was observed when LPS was added at a concentration of 0.1 μg/ml and 0.5 μg/ml.

Following staining with an annexin V/PI apoptosis kit, living cells demonstrated little or no fluorescence, and apoptotic cells exhibited green fluorescence. Necrotic cells exhibited both red and green fluorescence. As shown in Fig. 2A, the cells show weak green fluorescence, and no red fluorescence is found in the control groups. The fluorescence intensity of the 0.5 μg/ml groups was similar to that of the control group. However, the number of cells with strongly green fluorescence increased with the increasing concentrations of LPS (1–10 μg/ml). This indicates that apoptosis was more prevalent at high concentrations of LPS.

The results of Hoechst 33258 staining were consistent with the findings observed in a previous section. As shown in Fig. 2B, normal STC-1 cells exhibited weak fluorescence. Following LPS treatment, the cells demonstrated a brighter fluorescence, and typical apoptotic characteristics, such as nuclear condensation, were observed in cells treated with 1 or 10 μg/ml LPS.

The lower right quadrant of each plot indicates the percentage of early apoptotic cells that are only annexin V-FITC-positive. The upper right quadrant shows late apoptotic cells or necrotic cells with positive staining for annexin V-FITC and PI. In order to avoid interference from necrotic cells, the apoptotic cells in this portion of the plot were not included in the calculations. The viable cells (annexin V-FITC- and PI-negative) are shown in the lower left quadrant. As shown in Fig. 3, there was a trend of increased apoptosis in the 1 and 10 μg/ml groups compared with the control groups (up to 4.5 and 6.4%, respectively; P<0.05). However, no significant difference was observed between the 0.5 μg/ml LPS group and the control group.

As measured by western blotting (Fig. 4), incubation with LPS (0.5, 1 and 10 μg/ml) for 24 h markedly reduced the expression of Bcl-2 protein, to 75 and 45% (compared with the controls) in the 1 and 10 μg/ml LPS groups, respectively. The increased expression level of Bax protein paralleled the decrease in the expression level of Bcl-2 protein. However, Bax protein expression increased 2.2 and 3.0 times in STC-1 cells following treatment with 1 and 10 μg/ml LPS, respectively (controls are set as 100%). Furthermore, higher concentrations of LPS led to the increased cleavage of caspase-3. The levels of caspase-3 increased 2.0 and 2.6 times following treatment with 1 and 10 μg/ml of LPS, respectively. There were no significant changes in the expression of any of the three proteins in the 0.5 μg/ml LPS group.