Cell cycle analysis
In this study, propidium iodide (PI) (Becton Dickinson, USA) was used to
stain nuclear DNA of tumor cells to identify cell cycle changes. A549
cells were cultured for 24 hours after exposure to the compound NCTD at
concentrations of 0, 5, 10, 20, and 40 μM. Approximately 1 x 105 cells
were collected and fixed in 70% ice-cold ethanol overnight. The fixed
cells were incubated with 15 units / ml of RNase I and 100 μg / ml of
propidium iodide for 15 minutes. Cellular DNA content was measured by
flow cytometry. Data results were statistically analyzed by Cell Quest
software (FCM, Becton Dickinson, USA).
Measurements
of mitochondrial membrane potential (ΔΨm)
JC-1 dye (Beyotime Co, Hangzhou, China) was used to detect changes in
mitochondrial membrane potential (ΔΨm) of tumor cells, and this dye
indicated mitochondrial depolarization by changing the fluorescence
intensity from high red to low green. A549 cells were seeded in 6-well
plates, adjusted to a cell density of 1 × 105 cells / well, and exposed
to compounds NCTD at final concentrations of 0, 5, 10, 20 and 40 μM for
24 hours. Collect the cells and test them on a flow cytometer or spread
the cells on a glass slide. Images were acquired under a fluorescence
microscope (DFC480; Leica Microsystems, Wetzlar, Germany)..
Western
blot assays
After
treating A549 cells with different concentrations of 0, 5, 10, 20, and
40 μMNCTD for 24 hours, the cells were collected, and the concentration
of each group of proteins was measured after adding lysate for 30
minutes. The whole process was performed on ice. An equal amount of
protein was calculated based on the protein concentration. The volume of
each group was added to a 10% SDS-PAGE gel for electrophoresis, and
then the protein was transferred to a PVDF membrane. Block the membrane
with 5% skimmed milk powder / TBST for 100 minutes at room temperature,
and then react with the appropriate antibodies to be tested for diluted
primary antibodies Bcl-2, Bcl-xL, Mcl-1, caspas-3 / 8/8, PARP , AMPK,
p-AMPK, pc-Jun, p-Akt, Akt, c-Jun, JNK, ULK1, p-JNK, mTOR, Beclin-1,
LC3, p62, cyclinA, cyclinB2, cyclinD1, cyclinD3, p21, Cdc2 And the
internal reference GAPDH (diluted 1: 1000 in blocking buffer) at 4 ° C
overnight, remove the membrane from the primary antibody the next day,
wash 3 times in TBST for 10 minutes each time, and then peroxidize with
horseradish The enzyme-conjugated secondary antibody was incubated for 1
hour at room temperature. Wash the photos before the method, add the
developer Super Signal west pico (Thermo) together into the luminometer,
and visualize the protein.