Reagents
Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Nrf2 antibody (cat. no. sc-13032) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-β-actin antibody (cat. no. 4967) and anti-rabbit horseradish peroxidase (HRP)-linked IgG antibody (cat. no. 7074) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Ambion's Silencer® Select Pre-designed siRNA against the gene encoding Nrf2 (ID: s9492) and Silencer® Select Negative Control 1 siRNA were purchased from Life Technologies Corporation; Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
Cell culture
The A549 lung cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). A549 cells were maintained at 37°C in a humidified 5% CO2 atmosphere and cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 10% heat-inactivated FBS (Japan Bioserum Co., Ltd., Nagoya, Japan).
siRNA transfection
A549 cells were transfected with target or control siRNA using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.), as previously reported (17). The final concentration of siRNAs in the medium was 10 nM. After incubating the cells with the medium containing siRNAs for 48 h, transfected cells were collected and used for subsequent analyses.
In vitro irradiation
The cells were irradiated (150 kVp, 20 mA, 0.5-mm Al and 0.3-mm Cu filters) using an X-ray generator (MBR-1520R-3; Hitachi Medical Corporation, Tokyo, Japan) at a distance of 45 cm from the focus and a dose rate of 1.00–1.05 Gy/min.
Clonogenic survival assay
To examine the radiosensitivity, the cells were seeded on 60-mm diameter culture dishes (Iwaki, Chiba, Japan) and cultured overnight. After culturing for 6 h, the cells were exposed to X-ray radiation and incubated for the next 8–11 days. Next, the cells were fixed with methanol and stained with Giemsa solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Colonies containing >50 cells were counted.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis
Protein preparation and determination of the protein concentration were performed as reported previously (18). SDS-PAGE and western blot analysis were performed as reported previously (19). The following concentrations of primary antibodies were used: Anti-Nrf2 antibody (dilution, 1:3,000) and anti-actin antibody (dilution, 1:4,000). The secondary antibody used was HRP-linked anti-rabbit IgG antibody (dilution, 1:10,000). Antigens were visualized using the ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences, Chalfont, UK). Blots were stripped using a commercially available stripping solution (Wako Pure Chemical Industries, Ltd.).
Medium transfer experiments
The schematic for medium transfer experiments is shown in Fig. 1. Approximately 2.4×105 transfected cells were seeded onto 35-mm culture dishes and cultured for 5 h to promote their adherence to the dish. The cells were then exposed to X-rays, cultured for 24 h, and the cell conditioned medium was then collected by centrifugation (1,000 rpm for 5 min at room temperature). After centrifugation, the supernatant was collected and filtered using a 0.45-mm syringe filter (2053-025; Iwaki) to remove cells and debris. The filtrated cell conditioned medium (hereafter referred to as ICCM) was used for subsequent experiments.
One day before collecting the ICCM, approximately 6.0×104 cells were seeded onto 35-mm culture dishes for cell death analysis, or in a 12-well plate (BD Falcon) (100 or 120 cells) for colony assay, and cultured overnight to allow for their adherence to the dish. On the following day, the medium was aspirated and ICCM was added to the 35-mm culture dishes or to the 12-well plate. After 3 days of culturing, the cells that were seeded in the 35-mm culture dishes were collected using 0.1% trypsin-ethylene diamine tetraacetic acid (Gibco; Thermo Fisher Scientific, Inc.), and the number of viable cells was counted using the trypan blue dye exclusion assay. Finally, the harvested cells were used to perform the cell death analysis.
The cells that were seeded in the 12-well plates for colony assay were incubated for 8–10 days. The cells were then fixed with methanol and stained with Giemsa solution. Colonies containing >50 cells were counted.
Cell death analysis
Cell death was analyzed using Annexin V-FITC (BioLegend, Inc., San Diego, CA, USA), PI, and Annexin V binding buffer (BioLegend, Inc.), as reported previously (20). Stained cells were analyzed by performing flow cytometry (Cytomics FC500; Beckman Coulter, Inc., Brea, CA, USA). In the Annexin V/PI quadrant gating, Annexin V−/PI−, Annexin V+/PI−, and Annexin V+/PI+ were used to identify the fraction of viable cells, early apoptotic cells, and late apoptotic/necrotic cells, respectively.
Statistical analysis
Data are presented as mean ± standard deviation. Comparisons between control and experimental groups were performed using a two-sided Student's t-test or a two-sided Mann-Whitney U-test depending on the data distribution. Non-parametric multiple data were analyzed using the Kruskal-Wallis test followed by the Steel test. Differences were considered significant at P<0.05. All statistical analyses were performed using Excel 2016 software (Microsoft Corporation, Redmond, WA, USA), with an add-on software Statcel 4 (The Publisher OMS Ltd., Tokyo, Japan).