The MG63 osteosarcoma cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). RPMI-1640 medium was purchased from Gibco-BRL (Rockville, MD, USA); BafA1 was purchased from Biovision (Shanghai, China); L-glutamine was obtained from Sigma (St. Louis, MO, USA); and antibodies against p53, p62, Beclin1 and microtubule-associated protein 1 light chain 3 (LC3) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

BafA1 was diluted in dimethylsulfoxide (DMSO) to a final working concentration of 1 μmol/l and DMSO was used as the vehicle control.

MG63 cells were cultured in RPMI-1640 with 10% fetal bovine serum and 4 mmol/l L-glutamine. For all assays, the cells were cultured at 37°C in humidified incubators with 5% CO₂ and 95% air. The cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Labotatories, Kumamoto, Japan) assay. Cells were plated in 96-well plates at a density of 7×10⁴ cells/well and treated with BafA1 for 6, 12 or 24 h. Cell viability was subsequently assessed by the addition of 10 μl CCK-8 solution to each well, 24 h after BafA1 treatment. Following incubation with CCK-8 for 4 h, the optical absorbance at 570 nm was measured. Each experiment was performed in triplicate (13).

The ΔΦ was determined using the KeyGEN Mitochondrial Membrane Sensor kit (KeyGEN, Nanjing, China) according to the manufacturer’s instructions. Cells were first treated with BafA1, harvested and then washed three times with 5 ml phosphate-buffered saline (PBS), prior to centrifugation and aspiration. The cells were then re-suspended in 0.5 ml diluted MitoSensor reagent (1 μmol/m1 in incubation buffer; Becton-Dickinson, Heidelberg, Germany). Following incubation with the fluorescent probe JC-1 for 20–30 min, the cells were washed in 0.2 ml incubation buffer and resuspended in 40 μl incubation buffer, prior to re-washing and re-suspension in 1 ml PBS. The cells were then analyzed by flow cytometry (FACScan; Becton-Dickinson).

Cells treated with BafA1 for 6–24 h were harvested and washed three times with 5 ml PBS/0.1% fetal calf serum wash buffer, centrifuged and aspirated. The cells were then re-suspended in wash buffer containing 20 μg/ml propidium iodide (PI), 500 μg/ml RNase and 0.03% Nonidet P-40 and subsequently analyzed by flow cytometry. The percentage of apoptotic cells was taken as the percentage of cells with a lower DNA content than that of cells in the G₀-G₁ phase in the PI intensity-area histogram plot (14).

Cells were cultured for 24–48 h prior to lysis in buffer containing 50 mmol/l Tris-HCl (pH 8.0), 150 mmol/l NaCl, 1% (v/v) Triton X-100 and a protease inhibitor cocktail (1:100 dilution; Sigma, Shanghai, China). The protein concentration was determined using Bradford reagent. The proteins were then separated by electrophoresis using 8.5% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were subsequently exposed to anti-Beclin 1 (1:1,000), -p53 (1:2,000), -p62 (1:1,000) and -LC3 (1:1,000) antibodies and incubated at 4°C overnight, prior to exposure to horseradish peroxidase-conjugated secondary antibody (1:3,000) for 1 h at room temperature. β-actin (1:5,000; Sigma) was used as a loading control. Membranes were developed using an enhanced chemiluminescence detection system (Denville Scientific, Inc., Plainfield, NJ, USA) and exposed to X-ray films (15).

Following treatment with BafA1, the cells were fixed in ice-cold 2.5% glutaraldehyde in 0.1 M PBS and stored at 4°C, prior to further processing. The cells were post-fixed in 1% osmium tetroxide in ice-cold 2.5% glutaraldehyde in 0.1 M PBS and then dehydrated through an alcohol series prior to embedding in Epon™ 812 (Electron Microscopy Sciences, Hatfield, PA, USA). The cells were next sectioned using an ultramicrotome (Leica, Wetzlar, Germany). Finally, the sections (500 nm) were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy (Philips CM120; Philips, Eindhoven, The Netherlands).

The data are presented as the mean ± standard deviation. A Student’s t test was used for statistical analysis and a P-value of <0.05 was considered to indicate a statistically significant difference.

The CCK-8 assay revealed that the inhibition rate of MG63 cells treated with BafA1 (1 μmol/l) was significant1y higher than that of the controls (only vehicle used) (P<0.05). BafA1 inhibited the proliferation of the MG63 cells, with the rate of inhibition reaching 18±0.57% after 6 h of incubation. The inhibition rate increased to 39±2.82 and 56±3.91% by 12 and 24 h, respectively (Fig. 1).

MG63 cells showed a collapse in the Δψ after 6 h of exposure to BafA1 (1 μmol/l), with a maximum being reached by 24 h (Fig. 2). These data therefore indicated that BafA1 could induce mitochondrial dysfunction and apoptosis in MG63 cells.

The rate of apoptosis in MG63 cells was assessed by flow cytometry at 6, 12 and 24 h after exposure to BafA1 (1 μmol/l). BafA1-induced cellular apoptosis was evident after 6, 12 and 24 h of treatment (Fig. 3).

Western blot analysis was used to assess the effect of BafA1 on the expression of the apoptosis-related proteins Beclin 1 and p53. The results showed that the basal level of p53 protein in the untreated MG63 cells was low. Following incubation with BafA1 (1 μmol/l), the protein expression level of p53 and Beclin 1 was significantly increased 6–24 h after exposure (Fig. 4).

Western blot analysis was used to assay whether BafA1 (1 μmol/l) affected the expression of the autophagy-related proteins LC3-I, LC3-II and p62. The results showed that, prior to exposure of MG63 cells to BafA1, the basal levels of p62 and LC3 protein in MG63 cells were high. Following incubation for 6–24 h with BafA1 (1 μmol/l), the expression levels of p62 and LC3-I were significantly decreased, whereas the protein levels of LC3-II were increased (Fig. 5).

As shown in Fig. 6, transmission electron microscopy was used to identify the ultrastructural changes in MG63 cells following BafA1 (1 μmol/l) treatment. The control cells showed a round shape and the organelles, nuclei and chromatin had a normal appearance (Fig. 6A). By contrast, the BafA1-treated cells exhibited typical signs of apoptosis (Fig. 6B–D). The mitochondria exhibited vacuolization and swelling with a complete loss of cristae (Fig. 6C). Numerous isolated membranes, likely deriving from ribosome-free endoplasmic reticulum, were observed. These isolated membranes were elongated and curved, engulfing the cytoplasmic fraction and organelles (Fig. 6C). Prolongation of BafA1 treatment (1 μmol/l) resulted in apoptosis, as well as the loss of organelles and cytoplasm vacuolization (Fig. 6C and D).