Cells
Lung cancer A549 and NCI-H460 cell lines (Cell Bank, Chinese Academy of Sciences, Shanghai, China) were resuscitated and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 IU/ml streptomycin under 37˚C and 70% humidity in the presence of 5% CO2. The cell lines were passaged every three days and logarithmic growth cells were collected for experiments.
Logarithmic growth A549 and NCI-H460 cells (2×105) were seeded into 24-well plates containing antibiotics-free RPMI-1640 medium with 10% FBS. When the cells reached more than 70% confluency, they were transfected with scrambled RNA sequence (negative control (NC) group) and MUS81 interference sequence (siRNA-MUS81 group) (HanBio, Shanghai, China) according to the manufacturer’s manual. In a vial, 1.5 µl 20 pmol/ul NC or siR-MUS81 (HanBio, Shanghai, China) was mixed with 50 µL Opti Mem medium (Thermo Fisher Scientific, Waltham, MA, USA). In the other vial, 1 µL Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) was mixed with 50 µL Opti Mem medium. After five minutes, the mixtures in the two vials were combined and incubated for 20 minutes at room temperature. Then, the mixtures were added onto cells in respective groups. Six hours later, the medium was replaced with RPMI-1640 medium containing 10% FBS. After cultivation for 48 hours, the cells were collected for further assays.
CCK-8 assay
The cells were seeded into 96-well plates at a density of 1×104/well, and cultured in RPMI-1640 medium with 10% FBS. Cells were divided into propofol group and control group (3 replicates each). Propofol group was treated with 0.2, 0.5, 1 or 2 mM hydroxyurea (HU) for 24 h, and control group was treated with equal volumes of DMSO. After replacing the medium, the cells were cultured for additional 24 h. Then, 20 μl CCK-8 reagent (5 g/L; Beyotime, Shanghai, China) was added before incubation at 37˚C for 2 h. The absorbance of each well was measured at 490 nm for plotting cell proliferation curves.
Transwell assay
To test invasion ability, Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was thawed at 4°C overnight and diluted with serum-free RPMI-1640 medium (dilution 1:2). The mixture (50 μl) was evenly smeared into the upper chamber (Corning, Corning, NY, USA) and incubated at 37°C for 1 h before solidification. To test migration ability, Matrigel was not needed in the upper chamber. Cells (2 × 105) from each group were seeded into the upper chamber containing 200 μl serum-free RPMI-1640 medium. In addition, 600 μl RPMI-1640 medium supplemented with 10% FBS was added into the lower chamber. After 24 h, the chamber was removed and the cells in the upper chamber were wiped off. After being fixed with 4% formaldehyde for 10 min, the membrane was stained using Giemsa method for microscopic observation of 5 random fields (200×). The number of transwell cells was calculated for the evaluation of cell invasion and migration ability. All procedures were carried out on ice with pipetting tips being cooled at 4°C.
Flow cytometry
Cells in each group (1 × 106) were washed twice with precooled phosphate-buffered saline. BD Cycletest Plus DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to determine cell cycle according to the manufacturer’s manual. The result was analyzed using ModFit software version 3.2 (Verity Software House, Topsham, ME, USA).
Cells in each group (1 × 106) were washed with pre-cooled phosphate-buffered saline twice and subjected to flow cytometry using ANXN V FITC APOPTOSIS DTEC KIT I (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s manual to detect cell apoptotic rate. Cells with ANNEXIN V-positive values were early apoptotic cells, those with PI-positive values were necrotic cells, and those with double positive values were late apoptotic cells.
Laser confocal microscopy
NC group and propofol group of cells (1 × 106) were seeded onto petri-dishes (diameter, 6 cm) and incubated at 37˚C and under 5% CO2 for 24 h. Then, HU was used to stimulate the cells for 24 h. After discarding medium, the cells were washed with phosphate-buffered saline (PBS) for three times, and fixed with 4% formaldehyde for 10 min. After addition of 1.25 M glycine for neutralization, the fixing solution was discarded. After washing with cold PBS, the cells were perforated with 1% Triton X100 at room temperature for 5 min. After additional washing with PBS for 2 times, the cells were incubated with p-RPA32 at 4˚C overnight, followed by washing with PBS. Then, fluorescence secondary antibody was added for an incubation of 2 h. After additional washing with PBS for 3 times, the formation of foci was observed under a laser scanning confocal microscope (SP8; Leica, Wetzlar, Germany).
Western blotting
Cells in each group (1 × 106) were lysed with 1 ml precooled Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) for 30 min on ice. The mixture was centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was used to determine protein concentration by bicinchoninic acid (BCA) protein concentration determination kit (Beyotime, Shanghai, China). The samples were then mixed with 5× sodium dodecyl sulfate loading buffer before denaturation in boiling water bath for 10 min. Afterwards, the samples (20 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V. The resolved proteins were transferred to polyvinylidene difluoride membranes on ice (250 mA, 1 h) and blocked with 5% skimmed milk at room temperature for 1 h. Then, the membranes were incubated with rabbit anti-human ATR (1:1000), p-ATR (1:1000), RPA32 (1:1000), p-RPA32 (1:1000), MUS81 (1:1000), H3K27me3 (1:1000) (Cell Signaling Technology, Danvers, MA, UK) or mouse anti-human GAPDH monoclonal antibody (1:4000; Beyotime, Shanghai, China) polyclonal primary antibodies at 4°C overnight. After extensive washing with phosphate-buffered saline with Tween 20 (0.1%) for 5 times of 5 min, the membranes were incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology, Danvers, MA, UK) for 1 h at room temperature before washing with phosphate-buffered saline with Tween 20 for 5 times of 5 min. Then, the membrane was developed with enhanced chemiluminescence detection kit (Beyotime, Shanghai, China) for imaging. Image lab v3.0 software (Bio-Rad, Hercules, CA, USA) was used to acquire and analyze imaging signals. The relative contents of target proteins were expressed against GAPDH.
Statistical analysis
The results were analyzed using SPSS 18.0 statistical software (IBM, Armonk, NY, USA). The data were expressed as means ± standard deviations and underwent normality test. If the data fit normal distribution and the variance was homogeneous, multigroup measurement data were analyzed using one-way ANOVA and Dunnett’s test, while comparison between two groups was carried out using paired Student’s t-test. If the data were not normal distribution or the variance was not uniform, multigroup measurement data were analyzed using Kruskal-Wallis test and Tamhane's T2 or Dunnett's T3 method, while comparison between two groups was carried out using Mann-Whitney test. P < 0.05 indicated statistically significant differences.