The human hepatic cell line HepG2 (catalogue number TCHu72) was used in the present study. The cells were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained at 37°C in a cell culture incubator with a humidified atmosphere and 5% CO2. DIOS powder was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) to create a stock at a concentration of 10 mg/ml, which was subsequently diluted in culture medium to the corresponding concentration for subsequent experiments. DMSO was used as the vehicle control in the experiments.

Cells were seeded in 96-well plates at a density of 5,000–10,000 cells per well. After 24 h of treatment, DIOS (four replicates in each group) was added at different concentrations for various time periods. Then, 20 µl MTT solution (5 mg/ml) was added to each well, and the plates were incubated at 37°C for 4 h. The medium was removed, and 150 µl DMSO was added to dissolve the formazan crystals. The absorbance at 492 nm was measured using a spectrophotometer.

Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis detection kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's protocol. Cells were seeded in 6-well plates at a density of ~5×105 cells/ml. The cells were treated with DIOS at different concentrations (0, 5 and 10 µg/ml) and incubated at 37°C for 24 h. Subsequently, the cells were harvested and washed with PBS three times. Cells were resuspended in Annexin V binding buffer and labeled with the above Annexin V-FITC/PI apoptosis detection kit, and then analyzed by flow cytometry.

Antibodies specific for the following proteins were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and used at a 1:1,000 dilution: Notch3/cleaved Notch3 (#5276), NF-κB (#8242), p53 (#2527), PARP (#9532), IκB kinase (IKK)α (#11930), IKKβ (#8943) and GAPDH (#2118). Horseradish peroxidase-conjugated secondary antibodies included anti-rabbit (#E030120-01) anti-mouse (#E030110-02) antibodies at a dilution of 1:3,000 in TBST and were obtained from EarthOx Life Sciences (Millbrae, CA, USA). Following protein transferal, the membrane was blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies at 4̊C overnight. The membranes were washed three times for 5 min each time to remove unbinding antibodies and then incubated with the secondary antibodies at room temperature for 1–2 h. The membranes were washed again three times (5 min each time). Enhanced chemiluminescence buffer was applied to produce the protein signal and visualized using an x-ray film. The cells were treated with DIOS at different concentrations (0, 5 and 10 µg/ml) and incubated for 24 h at 37°C. Protein samples were treated with radioimmunoprecipitation assay cell lysis buffer (final phenylmethylsulfonyl fluoride concentration, 1 mM). Western blots were performed as previously described (23).

Cells (1×105) were plated in IF dishes (NEST Biotechnology, Wuxi, China) and treated with DIOS. Then, the cells were fixed in paraformaldehyde and permeabilized with 0.01% Triton X-100. Next, the cells were incubated with primary antibodies against p53 (#2527) and Notch3 (#5276) [1:500 dilution with 5% bovine serum albumin (BSA) in PBS; Cell Signaling Technology, Inc.] at 4°C overnight upon blocking the cells with 5% BSA dilution in PBS. DyLight™ 649-conjugated secondary antibodies (#009-490-006; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) were applied at a dilution of 1:500 at room temperature for 1 h. The specimens were monitored using a TCP SP5 laser microscope (Leica Microsystems GmbH, Wetzlar, Germany).

A specific small hairpin (sh)RNA targeting the Notch3 gene and a control shRNA (synthesized by Sangon Biotech Co., Ltd., Shanghai, China) were constructed into lenti-shRNA vectors, which were purchased from (GeneChem Co., Ltd., Shanghai, China). Upon virus production, these virus were transfected into HepG2 cell lines. Notch3 stable-depleted cells were obtained after 7 days of selection with puromycin. The cells were seeded in 6-cm plates for subsequent experiments.

Three groups of cells (HepG2 control, shRNA-vector-treated and shRNA-Notch3-treated cells) were plated in 6-well plates at ~1,000 cells/well and incubated for 7 days at 37°C. The medium was then removed and the cells were washed once with water. The clones were stained with crystal violet for 30 min and images were captured of the air-dried plate.

The data were analyzed with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA), and one-way analysis of variance was performed to compare differences between groups. All results shown represent means ± standard deviation from triplicate experiments performed in a parallel manner, unless otherwise indicated. All figures shown in the present study were obtained from at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

HepG2 cells were seeded at a density of 3×105 cells/cm2 12 h prior to DIOS treatment. The cells were exposed to three DIOS concentrations (0, 5 and 10 µg/ml), and DMSO served as the drug vehicle. The data indicated that DIOS induced cell apoptosis and inhibited cell proliferation in HepG2 cells in a dose- and time-dependent manner (Fig. 1A and B). Under light microscopy, the number of normal cells was significantly reduced, while the number of dead cells increased with time or dose. MTT analysis was used to evaluate the inhibitory effects and cytotoxicity of DIOS treatment in HepG2 cells. The data indicated that the cytotoxicity of DIOS increased in a concentration-dependent manner. The inhibitory concentration 50 of DIOS in HepG2 cells was 25 µg/ml for 24-h treatment (Fig. 1C).

Annexin V-FITC/PI double staining assay was performed to detect cell apoptosis in HepG2 cells subjected to DIOS treatment. The results indicate that DIOS could significantly induce cell apoptosis in HepG2 cells (P<0.05; Fig. 2A and B). IF indicated that p53 was upregulated following DIOS exposure by p53-specific antibody-mediated IF staining (Fig. 2C). By using western blotting, it was observed that the levels of p53 protein and its downstream target proteins were upregulated, while the levels of another anti-apoptotic protein, Bcl-2, were decreased (Fig. 2D).

HepG2 cells were treated with various concentrations of DIOS for 24 h. Notch3 and cleaved Notch3 protein levels were determined by western blotting, and it was noticed that Notch3, cleaved Notch3 and NF-κB were downregulated following DIOS exposure (Fig. 3A). It was confirmed that Notch3 expression was downregulated using IF staining (Fig. 3B). It was demonstrated that the inhibition of NF-κB resulted from the inactivation of IKKα and IKKβ (Fig. 3C). These results indicated that DIOS could regulate the Notch3 signaling pathway by modulating the Notch3 receptor level and Notch3 activation processes.

Specific small interfering RNAs for Notch3 and p53 were applied for gene knockdown (Fig. 4A and B). Deletion of Notch3 inhibited p53 expression, while knockdown of p53 upregulated Notch3 receptor content and enhanced the release of cleaved Notch3. These results indicated that p53 negatively regulated the Notch3 pathway, while Notch3 promoted p53 expression, which suggests that there is negative feedback regulation between Notch3 and p53. Additionally, reduced expression of Notch3 led to a lower expression of NF-κB and p53. p53 knockdown induced Notch3 receptor upregulation, release of cleaved Notch3 and downregulation of NF-κB (Fig. 4B). It was identified that Notch3 inhibition enhanced the DIOS-caused damage of clone forming ability in HepG2 cells (Fig. 4C). Evaluation of DIOS-treated HepG2 cells, Notch3-control HepG2 cells and Notch3-knockdown HepG2 cells revealed that downregulation of Notch3 rescues the p53 upregulation induced by DIOS treatment. Deletion of Notch3 enhanced the reduction in NF-κB levels that was mediated by DIOS exposure. The apoptotic proteins Bax and poly (ADP-ribose) polymerase (PARP) were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated (Fig. 4D). The data indicated that DIOS treatment could inhibit the NF-κB pathway and activate the apoptosis pathway.