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.