Lung adenocarcinoma A549 cells were kindly donated by The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing a mixture of 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and 1% streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO₂, as previously described (9). A549 cells at the logarithmic growth phase were seeded (6x10⁵ cells/well) into six-well culture dishes. Once the cells have adhered, resveratrol (Sigma-Aldrich; Merck KGaA) was dissolved in dimethylsulfoxide (Sigma-Aldrich; Merck KGaA) to produce a stock solution of 10,000 µM, and stored at -20˚C. Different concentrations of resveratrol (0, 10, 50, 100, 150 and 200 µmol/l) were added into each dish and incubated for 24 h at 37˚C, and the resveratrol-containing medium was removed with a pipette. The same resveratrol treatment groups were prepared and irradiated with 0, 2, 4 and 6 Gy X-rays (room temperature, 6 MV X-rays, 300 cGy/min, the distance from the source to the specimen was 100 cm and the size of the irradiation field was 20x20 cm). The culture was continued for 4 h at 37˚C after irradiation for the following experiments.

Cell counting was performed to perform an approximate evaluation of cell damage from irradiation and resveratrol treatment. In total, 0.8x10⁶ A549 cells were seeded into 60-mm culture dish to conduct the following experiment. After receiving corresponding experimental treatments, the culture medium was replaced to wash out dead cells and the remaining A549 cells growing on the bottom of the 60-mm culture dish were placed under a light microscope (magnification, x40) to check the overall growth state. In order to quantify the cell number, A549 cells were resuspended in 1X PBS and 100 µl cell suspension was added to the corresponding chip, which was inserted into the cell counting machine (Scepter2.0; Sigma-Aldrich; Merck KGaA) in order to calculate the concentration of the remaining cells within each 100-µl A549 cell suspension. Together, the data were used to calculate the total remaining A549 cell number within an entire 50-mm culture dish.

CCK-8 assay was performed to check the cell viability after respective experimental treatments. A549 cells in the logarithmic growth phase were dissociated with trypsin and 5x10³ cells/well were added to a 96-well plate. Once the cells had adhered to the well, they were divided into different groups and treated with various concentrations of resveratrol and different doses of irradiation, as aforementioned. After incubation at 5% CO₂ and 37˚C for 4 h, 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well. After 2 h of incubation, the optical density of each well was measured at a wavelength of 450 nm on an enzyme-linked immunosorbent detector (Beyotime Institute of Biotechnology).

A549 cells (2.5x10⁵ cells/ml) were seeded onto each well of a 24-well plate and incubated for 24 h. After treatment with different doses of resveratrol (0, 10, 50, 100, 150 and 200 μM) combined with different doses of irradiation (0, 2, 4 and 6 Gy), the culture medium was removed from each well and the cells were washed twice with PBS at room temperature. Rhodamine 123 powder (Dalian Meilun Biology Technology Co., Ltd.) was prepared into a 1 mg/ml stock solution with methanol and stored at -20˚C. The cells were incubated with 5 µg/ml rhodamine 123 for 30 min in a 5% CO₂ cell incubator at room temperature, then washed with PBS two or three times, and finally 0.5 ml PBS was added to each well. The plate was placed under a fluorescent microscope (Nikon M50; Nikon Corporation) with parameters initially set to detect fluorescence of rhodamine 123 (480/40 excitation filter, 505 long pass dichroic mirror and 535/40 emission filter; magnification, x20) and red fluorescence dots indicated mitochondria inside the cells (10). Briefly, once a clear visual field with 200-300 cells is achieved, images were captured at five different and qualified visual fields randomly within one well for each experimental group. The fluorescence intensity was reversely associated with Δψm and mean fluorescent intensity within an image was analyzed using ImageJ 1.8.0 software (National Institutes of Health).

RIPA buffer (Beyotime Institute of Biotechnology) was used to extract total cell protein in different dose groups, and the protein concentration was determined spectrophotometrically using a BCA assay (Beyotime Institute of Biotechnology). After boiling and denaturing, 30 µg proteins were separated by 10% SDS-PAGE and transferred to a PVDF film (EMD Millipore). TBS-T (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) containing 5% skim milk was used to block the membranes for 2 h room temperature. The following primary antibodies (diluted 1:1,000; all ProteinTech Group, Inc.), were added and incubated at 4˚C overnight: Rabbit anti-human STIM1 (cat. no. 11565-1-AP), STIM2 (cat. no. 21192-1-AP), mouse anti-human Orai1 (cat. no. 66223-1-lg), Orai2 (cat. no. 20592-1-AP) and Orai3 (cat. no. 25766-1-AP). β-actin (cat. no. 66009-1-Ig) were used as an internal control. The following day, TBST was used to wash the membrane for 10 min, and HRP-labeled anti-rabbit or anti-mouse antibodies were added (1:2,000; cat. nos. SA00001-1 and 11565-1-AP, respectively; ProteinTech Group, Inc.). After 1 h incubation at room temperature, the membranes were washed for 10 min three times with PBS-T, and the bands were visualized using an ECL development gel imaging system (ImageQuant LAS 500; Cytiva). Each experiment was repeated at least three times. Densitometry for each graph was conducted using ImageJ 1.8.0 software (National Institutes of Health).

After experimental treatment, a round coverslip was placed on the bottom of a 12-well plate using tweezers, and 150 µl 100 µg/ml polylysine was added to the middle of each coverslip. The polylysine was then removed by pipette after 10 min. A total of 4x10⁴ cells were seeded on circular coverslips. Following incubation for 8 h with culture medium, 10 µmol/l Fluo-8 AM (cat. no. ab112129; Abcam), a fluorescent probe for intracellular Ca²⁺ ([Ca²⁺]i), was added and incubated at 37˚C for 30 min. The sample was washed twice with Ca²⁺-free PBS buffer (standard external solution including 10 µM HEPES, 120 µM NaCl, 5.4 µM KCl, 1 µM MgCl₂ and 10 µM glucose; pH 7.4) and calcium fluorescence was detected under a fluorescent microscope with MetaFluor software 7.0 (Molecular Devices, LLC) (magnification, x200). Briefly, stained cells were initially observed under white light, by adjusting the focus, opening the fluorescence and detecting the cells in darkness. The software tools were used to capture each cell outline within the visual field and their fluorescence intensity was recorded. When the fluorescence intensity curve become stable for 3 min, Ca²⁺ release was induced by 2 µmol/l thapsigargin (cat. no 12758; Cell Signaling Technologies, Inc.) administration and left for another 5 min until the fluorescent curve become stable. Extracellular Ca²⁺ solution (cat. no. c1016; Sigma-Aldrich; Merck KGaA; 1 mmol/l) was then added to induce calcium influx with anther fluorescent peak and left for 5 min until the fluorescent curve became stable again. The process was observed under a fluorescence microscope (Nikon M50; Nikon Corporation) at 488 nm excitation and 515 nm emission wavelengths (magnification, x200). The change in [Ca²⁺]i was compared with the fluorescence intensity (F0) before adding extracellular Ca²⁺. SOCE was demonstrated as the ratio of peak intensity (F1)/F0 and the intensity of each fluorescence data was an average of 20-30 cells (11). Data were collected and analyzed with MetaFluor software7.0 (Molecular Devices LLC).

The Orai1 cDNA sequence was inserted into a pCMVPuro01 expression vector (Guangzhou Sinogen Pharmaceutical Co., Ltd.). The forward and reverse primer sequences for constructing the vectors were 5'-CTAGTCTAGAATGCATCCGGAGCCCGCCC-3' and 5'-TCCTTCGAACTAGGCATAGTGGCTGCCGG-3'. The STIM1 cDNA sequence was also inserted into a pCMVPuro01 expression vector (Guangzhou Sinogen Pharmaceutical Co., Ltd.). The forward and reverse primer sequences for constructing the vectors were 5'-AAGAGTCTACCGAAGCAG-3' and 5'-GTGCTATGTTTCACTGTTGG-3'. Empty vectors were used as a negative control. Briefly, an appropriate number of cells (4x10⁴) were seeded into each well (12-well dish) with 1 ml medium and incubated at 37˚C and 5% CO₂ until the cells reached 80-90% confluence for transfection. Orai1 overexpression vector (100 pmol) and/or STIM1 overexpression vector (100 pmol) were added to 100 µl Opti-MEM I reducing serum medium (Invitrogen; Thermo Fisher Scientific, Inc.) within an Eppendorf tube, and 5 µl Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was added to the same tube. The tube was gently agitated and incubated at room temperature for 25 min to form Lipofectamine complexes. The old medium was aspirated, and 1 ml serum-free medium was added. The Lipofectamine complex (100 µl) was added to each well and mixed gently. The cells were incubated at 37˚C and 5% CO₂ for 8 h and the serum-free medium was replaced with normal medium. Transfection efficiency was assessed via western blotting after 48 h.

All experiments were repeated independently at least three times. Data are presented as the mean ± SD. Multiple comparison analysis was conducted using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc.) and SPSS 19.0 (IBM Corp.).

A549 cells at the logarithmic growth phase were divided into different experimental groups, receiving a series of concentrations of resveratrol (0, 10, 50, 100, 150 and 200 µmol/l) combined with different doses (0, 2, 4 and 6 Gy) of X-ray irradiation. After experimental treatment, cell growth was observed, and cell numbers were counted and compared under a light microscope. The data showed that increased cell death and reduced cell numbers depended on resveratrol and irradiation dose. For each irradiation dose, compared with the 0 µM Res group, a significant cell number reduction was observed initially from 100 µM resveratrol treatment group. Among all the irradiation doses with 100 µM resveratrol, a significant cell number reduction was observed between 2 and 4Gy. Although combination of higher doses of irradiation and resveratrol also significantly reduced the cell number, based on the principle that the least but significant dosage might be the best, the combination of 4 Gy irradiation and 100 µM resveratrol could be used as an ideal treatment strategy for the following experiments (Fig. 1A). CCK-8 assay was further used to detect A549 cell viability in different dose groups. Similar to light microscopy results, irradiation promoted the reduction of cell viability, and resveratrol pretreatment enhanced this effect (Fig. 1B). The Δψm is a key parameter of mitochondrial function, and its decrease is often associated with apoptosis (25). Therefore, the fluorescent lipophilic cationic dye rhodamine 123 was used for Δψm detection in each group. As a positively charged dye, rhodamine 123 will accumulate in the mitochondria in an inverse proportion to the Δψm. The accumulation of fluorescent dyes in mitochondria was detected by confocal microscopy to evaluate Δψm to compare apoptosis (26). Fluorescent microscopy showed that both resveratrol and irradiation treatment reduced the Δψm of A549 cells in a dose-dependent manner and the combination of resveratrol and irradiation enhanced this effect (Fig. 2A and B), which was also consistent with aforementioned experiments (Fig. 1).

The aforementioned results indicated that resveratrol had the potential to be developed as an irradiation sensitizer. Thus, in order to further investigate the subcellular mechanism, the effects of resveratrol combined with irradiation treatment on thapsigargin-induced SOCE in lung adenocarcinoma A549 cells were detected using calcium imaging in four experimental groups: Blank control, 4 Gy irradiation, 100 µM resveratrol and 4 Gy irradiation + 100 µM resveratrol using Fluo-8, an intracellular calcium fluorescent probe. When the fluorescence intensity curve was detected to be stable, thapsigargin was utilized to cause calcium storage depletion, and the level of intracellular calcium rapidly increased and then decreased to normal levels. Subsequently, extracellular calcium was added to trigger SOCE, which demonstrated another instant increase in intracellular fluorescence (Fig. 3B). Based on the results, the peak level of fluorescence intensity was significantly reduced by resveratrol and this reduction was enhanced when combined with irradiation, indicating that SOCE was significantly inhibited by the addition of resveratrol (Fig. 3A and C).

STIM and Orai family members are the SOCCs mediating the SOCE process in non-excited cells, responsible for inflow, storage and consumption of calcium ions (13). Thus, western blotting was used to detect the expression of STIM1, STIM2, Orai1, Orai2 and Orai3 proteins in four experimental groups: Blank control, 4 Gy irradiation, 100 µM resveratrol and 4 Gy irradiation + 100 µM resveratrol. The results showed that compared with controls, resveratrol alone or resveratrol combined with irradiation treatment had no significant effect on the expression of STIM2, Orai2 and Orai3 in A549 cells but significantly suppressed the expression of STIM1 and Orai1. Among the four groups, combined treatment exerted the strongest effects (Fig. 4A and B). To some extent, these results indicated that resveratrol-sensitized irradiation damage in A549 cells may be associated with STIM1 and Orai1 downregulation that leads to SOCE suppression.

Downregulation of SOCE was associated with apoptosis in various cell types, including osteosarcoma, skin cancer and renal cancer (27-29). According to the aforementioned results, it was found that resveratrol can inhibit SOCE by downregulating the expression of STIM1 and Orai1, further leading to A549 cell death. To further verify this result, STIM1 and Orai1 were overexpressed, following resveratrol treatment and irradiation, in order to observe whether the recovery of STIM1 and Orai1 could restore SOCE and cell death in A549 cells. Western blotting images (Fig. S1A and B) and corresponding analysis (Fig. S1C and D) demonstrated that the vectors were transfected successfully. The expression levels of STIM1 and Orai1 proteins, following respective or combined treatment with Orai1 and STIM1 overexpression vectors after administration of both resveratrol and irradiation, were significantly increased compared with the pure combined treatment group with resveratrol and irradiation (Fig. 5A and B). Calcium imaging (Fig. 6A and B) and corresponding analysis of SOCE (Fig. 6C) in each group were partly consistent with our hypothesis. Although the results were not statistically significant, the SOCE of the combined resveratrol and irradiation treatment group was partly restored after adding both STIM1 and Orai1 overexpression vectors compared with single treatment groups. Additionally, CCK-8 results indicated that cell viability of the resveratrol combined with irradiation treatment group was partly restored after adding both STIM1 and Orai1 overexpression vectors compared with single treatment groups (Fig. 5C). Based on the aforementioned findings, the role of resveratrol in sensitizing A549 cells to irradiation treatment may be associated with downregulating the expression of STIM1 and Orai1, inhibiting the SOCE process and triggering cell death.