A549 lung cancer cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM/F-12 (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 10% heat-inactivated fetal bovine serum (HyClone; GE Healthcare Life Sciences) and 100 U/ml penicillin and streptomycin. Cells were grown in 25 cm² culture flasks at 37°C in a humidified atmosphere containing 5% CO₂. LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in PBS. Pretreatment with 10 µg/ml TP [dissolved in dimethyl sulfoxide (DMSO)] (Sigma-Aldrich; Merck KGaA) lasted for 48 h, and then there was an interval of 1 h prior to LPS treatment.

Cell viability was determined using the colorimetric MTT assay (Sigma-Aldrich; Merck KGaA). Briefly, A549 cells were seeded in 96-well tissue culture plates at a density of 5×10⁴ cells/well. After 24 h, cells were treated with 0.5, 1 or 10 µg/ml LPS for 48 h or 10 µg/ml LPS for 12, 24 or 48 h, and then cultured in fresh medium containing 0.5 mg/ml MTT for 4 h at 37°C. Subsequently, the formazan crystals that formed were dissolved in DMSO and the absorbance was determined at 550 nm.

A549 cells were seeded in 6-well tissue culture plates at a density of 1×10⁵ cells/well. When they had reached 70–80% confluence, cells were cultured for 16 h in serum-free DMEM/F-12. Following treatment with 10 µg/ml LPS for 48 h at 37°C, cells were incubated with 1 mM dichlorodihydrofluorescein diacetate for 40 min at 37°C in the dark. Cells were harvested and re-suspended in PBS. The relative fluorescence intensity was determined using a flow cytometer a BD FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA) and data was analyzed using the ModFit software version 4.1 (Verity Software House, Inc., Topsham, ME, USA).

Cellular apoptosis was determined using the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences), according to the manufacturer's protocol. Following treatment with 10 µg/ml LPS for 48 h at 37°C, cells were collected in a 5 ml culture tube and washed twice with ice-cold PBS. Cells were subsequently re-suspended in binding buffer and transferred to a new 5 ml culture tube. Annexin V-FITC (5 µl) and propidium iodide (5 µl) were added, and cells were incubated at room temperature for 15 min in the dark. Finally, 400 µl binding buffer was added and apoptotic cells were analyzed using a FC500 flow cytometry instrument equipped with CXP 2.0 software (Beckman Coulter, Bethesda, MA, USA) within 1 h.

Following treatment with 10 µg/ml LPS for 48 h at 37°C, cells were washed with ice-cold PBS three times and then fixed with 4% formaldehyde in PBS for 15 min (37°C). Subsequently, cells were stained with Hoechst 33258 (10 µg/ml) at 37°C for 5 min and washed three times with PBS. Finally, cells were observed under a fluorescent microscope (Olympus Corporation, Tokyo, Japan; magnification, ×40).

Total protein was extracted from cells using radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), according to the manufacturer's protocol. A bicinchoninic protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. Immunoblotting was performed as previously described (16). Primary antibodies against SIRT1 (cat. no., 9475; dilution, 1:1,000), p62 (cat. no., 23214; dilution, 1:1,000), acetyl-p53 (cat. no., 2570; dilution, 1:1,000), p53 (cat. no., 2524; dilution, 1:1,000), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax; cat. no., 5023; dilution, 1:1,000), Bcl-2 (cat. no., 2872; dilution, 1:1,000), and β-actin (cat. no., 4970; dilution, 1:4,000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). These antibodies were incubated with the membranes at 4°C overnight. Following three washes (10 min/wash) with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no., ZB-2306; Zhongshan Golden Bridge Biological Technology Co., Beijing, China) for 2 h at room temperature, and then washed three times (10 min/wash). The proteins were detected using enhanced chemiluminescence, according to the manufacturer's protocol (Merck KGaA, Darmstadt, Germany). ImageJ 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was applied to quantify the relative protein levels. β-actin was used as an internal control.

A549 cells were seeded at a density of 5×10⁵ cells/well in 6-well plates for 24 h. Transfection of a GFP-LC3-expressing plasmid (cat. no. 17-10230; Merck KGaA, Darmstadt, Germany) was performed using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. After 24 h, cells were treated with 10 µg/ml LPS or 10 ng/ml rapamycin (Rap; R8718, Sigma-Aldrich; Merck KGaA) for 48 h at 37°C, and incubated for 16 h. GFP-LC3-positive cells were observed using a confocal laser microscope (LSM700; Carl Zeiss AG, Oberkochen, Germany).

Samples were fixed with 2% glutaraldehyde-paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 12 h at 4°C and washed three times for 30 min with 0.1 M phosphate buffer. Subsequently, samples were incubated with 1% OsO₄ dissolved in 0.1 M phosphate buffer for 2 h, dehydrated in an ascending ethanol series (50–100%) and infiltrated with propylene oxide. The Poly/Bed^® 812 kit (Polysciences, Inc., Warrington, PA, USA) was used for resin embedding and polymerization was performed at 60°C in an electron microscope oven (TD-700; DOSAKA, Kyoto, Japan) for 24 h according to the manufacturer's protocol. Sections 350 nm in size were cut and stained using toluidine blue for 20 min at room temperature (for light microscopy) and 70 nm thin sections were double-stained using 7% uranyl acetate and lead citrate for 20 min at room temperature. Sections were cut using a Leica Ultracut UCT Ultra-microtome (Leica Microsystems GmbH, Wetzlar, Germany) and observed using a transmission electron microscope (JEM-1011; JEOL, Ltd., Tokyo, Japan) at an acceleration voltage of 80 kV.

Cells were transfected with small interfering RNA (siRNA) targeting si-SIRT1 (12241, Cell Signaling Technology, Inc., Danvers, MA, USA), or with negative control siRNA (NC; 5′-ACUAGUCGAUCUAUGUGUGAUATT-3′) (Shanghai GenePharma Co., Ltd., Shanghai, China) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at the final concentration of 20 nM, according to the manufacturer's protocol. In brief, A549 cells (1×10⁶ cells/well) were seeded in a 6-well plate with 2 ml RPMI-1640 medium (HyClone; GE Healthcare Life Sciences). At 60% confluence, si-SIRT1 or NC was mixed with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 20 min. Then, the mixture was added into each well at a final concentration of 20 nM for 48 h. Then, the cells were collected for further analysis.

Data are expressed as the mean ± standard error of the mean. Multiple comparisons were evaluated by analysis of variance followed by Tukey's multiple-comparison test. P<0.05 was considered to indicate a statistically significant difference.

A549 cells were treated with 0.5, 1 or 10 µg/ml LPS for 48 h, and cell viability was determined using the MTT assay. As presented in Fig. 1A, treatment with LPS suppressed the viability of A549 cells in a dose-dependent manner. A549 cells were also treated with 10 µg/ml LPS for 12, 24, 48 h. Notably, treatment with LPS inhibited the viability of A529 cells in a dose-dependent manner (Fig. 1B).

The effect of LPS treatment on the induction of apoptosis in A549 cells was investigated using flow cytometric analysis. LPS significantly increased the proportion of A549 apoptotic cells ~2.3-fold compared with the untreated control (Fig. 2A). Furthermore, Hoechst 33258 staining demonstrated increased numbers of apoptotic cells following treatment with LPS (Fig. 2B). ROS generation following LPS treatment was also examined. As presented in Fig. 2C, treatment with LPS significantly increased ROS generation in A549 cells in a time-dependent manner. Western blot analysis demonstrated that LPS treatment markedly decreased the protein level of SIRT1, whereas the acetylated and total p53 protein expression were increased. Additionally, the expression of the pro-apoptotic protein Bax was significantly induced, whereas the expression of Bcl-2 was suppressed (Fig. 2D). These results suggest that LPS treatment induces a pro-apoptotic effect in A549 cells.

The effect of LPS treatment on the induction of autophagy was also investigated. Rap was used as a positive control. Electron microscopy demonstrated that LPS induced autophagy in A549 cells in a similar pattern to that induced by Rap (Fig. 3A). Following GFP-LC3 transfection increased numbers of autophagic vesicles (autophagosomes) were observed in A549 cells treated with LPS or Rap (Fig. 3B). Western blot analysis indicated that treatment with LPS significantly decreased p62 protein expression and increased the LC3II/LC3I ratio (Fig. 3C).

A549 cells were treated with 10 µg/ml TP for 48 h and total protein was extracted from cells. As presented in Fig. 4A, treatment with TP significantly increased the protein expression of SIRT1. ROS generation following LPS treatment with or without 10 µg/ml TP was also examined. TP treatment significantly decreased ROS generation in LPS-treated A549 cells (Fig. 4B). Furthermore, the effect of TP on the induction of apoptosis in A549 cells treated with LPS was also investigated and it was demonstrated that treatment with TP reversed the LPS-induced apoptosis (Fig. 4C). Additionally, western blot analysis demonstrated that transfection with a small interfering RNA targeting SIRT1 inhibited the expression of SIRT1 even in the presence of TP. Furthermore, the expression levels of acetylated and total p53 were also reversed by TP treatment, whereas the protein expression of p62 and Bcl-2 was induced and the protein expression of Bax was suppressed. These results indicate that TP serves an anti-apoptotic role in A549 cells treated with LPS and that this effect is abolished by SIRT1 silencing (Fig. 4D).