Cell lines and culture conditions
Human lung cancer cell lines A549 and H1299 were purchased from Keygen Biotech (Nanjing, China). The radioresistant derivatives A549R and H1299R were generated by dose-gradient irradiation of the parental cells. All cells were maintained in RPMI-1640 medium (Gibco, New York, MD, USA) containing 10% fetal bovine serum at 37°C with 5% CO2 in a humidified incubator.
Dose-gradient irradiation
Irradiation was performed at a dose rate of 300 cGy/min at room temperature using a Varian 23 EX Clinac linear accelerator (Varian Medical Systems, Inc., Palo Alto, CA, USA). For the first irradiation, A549 and H1299 cells were grown to 60%–70% confluence and irradiated with 2 Gy of X-ray; the culture medium was replenished immediately after irradiation. When the cells reached the confluence of more than 80%, they were trypsinized and passaged. After two passages, the same irradiation and cell propagation procedure was performed. The procedure was further repeated with gradually increased radiation dose, and each dose was used twice. In total, the cells received 60 Gy of radiation (2 × 2 Gy, 2 × 4 Gy, 2 × 6 Gy, 2 × 8 Gy, and 2 × 10 Gy). The surviving cells were propagated and passaged for five or more generations before being used for other experiments.
Cell viability/proliferation assay with Cell Counting Kit-8
A Cell Counting Kit-8 (CCK-8) kit (Dojindo Laboratories, Kumamoto, Japan) was used to determine cell viability and proliferation after irradiation. Briefly, the cells were seeded in a 96-well plate (3000 cells/well, four replicates for each cell line) and incubated overnight. The cells were irradiated with five different doses (0, 2, 4, 6, and 8 Gy) and then incubated for further 48 h. The cells were replenished with a medium containing CCK-8 solution (10 μL CCK-8 in 100 μL medium) and incubated for another 2 h; then the absorbance at 450 nm was measured using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The survival rate of cells was calculated as the normalized absorbance to the non-irradiated controls.
Apoptosis detection
Cells were stained with an Annexin V-FITC detection kit (KeyGen, Nanjing, Jiangsu, China), following the manufacturer’s instructions, and analyzed with a BD FACScan system (BD Biosciences, San Jose, CA, USA). The graph was plotted using Flowjo 7.6.5 software (FLOWJO LLC, Ashland, KY, USA).
Plasmids and transfections
The pEGFP-LKB1, pEGFP-Ctrl, pshLKB1, and pshCtrl plasmids were constructed by GenePharma (Shanghai, China). Human LKB1 open reading frame was inserted in-frame with enhanced green fluorescent protein (EGFP) into the pEGFP-N1 vector to obtain the pEGFP-LKB1 vector. pGenesil-1 is a derivative of the pEGFP-C1 vector, which contains a human U6 promoter to drive short hairpin RNA (shRNA) expression. A DNA fragment encoding an shRNA against human LKB1 was inserted into the pGenesil-1 vector to obtain pshLKB1; the scrambled shRNA was also cloned into pGenesil-1 to obtain pshCtrl. The target sequence of LKB1 was 5′-GGTACTTCTGTCAGCTGATTG-3′, and the scrambled shRNA sequence was 5′-GTTCTCCGAACGTGTCACGTT-3′. Transient transfection was performed with Lipofectamine 2000 (Invitrogen, Shanghai, China), following the manufacturer’s instructions. Twenty-four hours after transfection, the cells were harvested for either Western blot analysis or further functional tests.
Western blotting and antibodies
Western blotting was performed as described previously [19]. The following primary antibodies were used: LKB1 (ab15095, 1:100) from Abcam (Cambridge, UK); E-cadherin (BS1097, 1:500), vimentin (BS1776, 1:500), β-actin (BS6007 M, 1:10,000), p-CHK2 (p-T68) (BS4043, 1:500), and γ-H2AX (p-S139) (BS4760, 1:500) from Bioworld Technology (Nanjing, Jiangsu, China); and SIK1 (51045-1-AP, 1:1000) and ZEB1 (21544-1-AP, 1:1000) from Proteintech Group (Wuhan, Hubei, China). All primary antibodies were incubated with the blot at 4°C overnight. The signals were detected with an Odyssey Infrared Imaging system (LI-COR, Lincoln, NE, USA). For quantification of the protein levels, the intensity of each strip was analyzed by Image J software (NIH, Bethesda, MD, USA). The average intensities of the proteins were normalized to β-actin. The relative protein levels are presented as mean ± standard deviation (SD).
Cell invasion assay
Invasion ability of the cells was determined using a modified two-chamber plate with a pore size of 8 μm. The transwell filter inserts were coated with Matrigel (BD Biosciences, New York, NJ, USA), and 5 × 104 cells were seeded in serum-free medium in the upper chamber. After incubation for 48 h at 37°C, cells in the upper chamber were carefully removed with a cotton swab, and the cells that had traversed the membrane were fixed in methanol and stained with crystal violet. The cells were counted under an inverted microscope and photographed. If transient transfection was performed prior to the assay, the cells were seeded 24 h after the transfection.
Cell migration assay
Migration assay was performed similarly to the invasion assay, with the following differences: the transwell filter inserts were not coated; 3 × 104 cells were seeded; and the incubation time was 24 h before fixation.
Wound healing assay
A wound was made by dragging a yellow pipette tip along the center of the plate. The distance between the cells bordering the wound was measured after 24 h. Images were taken with a digital camera under a phase-contrast microscope. If transient transfection was performed prior to the assay, the cells were wounded 24 h after the transfection.
Clonogenic cell survival assay
The cells were seeded in six-well plates (200, 400, 1000, 3000, and 5000 cells/well in triplicate, corresponding to the radiation dose of 0, 2, 4, 6, and 8 Gy, respectively). After overnight incubation, the cells were irradiated with the respective dose. The medium was replenished after the irradiation, and the cells were cultured for 14 days prior to formaldehyde fixation and crystal violet staining. The colonies with 50 or more cells were counted. The plating efficiency was calculated as the ratio of the colonies number to the plated cell number. The survival fraction was calculated as the normalized plating efficiency to the non-irradiated controls. Using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA), the survival curves were fitted to a linear-quadratic model to estimate the sensitizer enhancement ratio.
Statistical analysis
All statistical analyses were performed by using SPSS version 16.0 software (IBM, Chicago, IL, USA) except for those specially stated. All values are shown as mean ± SD. Student’s t test and one-way analysis of variance (ANOVA) were used to evaluate significance. P values less than 0.05 were considered statistically significant. All tests were two-tailed.