2.1. Drugs
Berberine(Ber) and formononetin were purchased from Shanghai Jinsui Biotechnology Co., Ltd. (Shanghai, China), and the purity was > 99 %. The powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a 200 mM stock solution, which was separated and stored at −20 °C in the dark. Roswell Park Memorial Institute (RPMI) 1640 medium was used to dilute the stock solution to prepare working concentrations. EGF(Meilun Bio) was dissolved in 20 μg/ml DMSO, and 1640 medium was used to dilute the working solution to 50 ng/ml. PD98059 (MCE) was dissolved in 50 mM DMSO, and 1640 medium was used to dilute the working solution to 50 μM. 3-Methyladenine(3-MA, MCE, Monmouth Junction, NJ, USA) was dissolved in 200 mM DMSO, and 1640 medium was used to dilute the working solution to 2 mM.Rapamycin (RAPA,MCE) was dissolved in 25 μM DMSO, and 1640 medium was used to dilute the working solution to 50 nM.
2.2. Cell culture
The human NPC cell line CNE1 , CNE2 were purchased from BeNa Culture Collection (Beijing, China). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin. Cells were cultured at 37 °C with 5% CO2.
2.3. Real-time monitoring of cell proliferation by real-time cellular analysis (RTCA)
The RTCA monitoring system uses cell index (CI) to reflect cell proliferation. The logarithmic growth phase cells were taken, digested, and resuspended into a single cell suspension, and 5 × 103 cells/well were seeded on the RTCA cell proliferation monitoring culture plate, and each group was set with three multiple wells.
2.4. CytationTM5 cell imaging multifunctional detection system
The cells are routinely digested and the cell suspension prepared. 5 × 103 cells/well were seeded in a 96-well plate, and the drug was added to the CytationTM5 cell imaging multifunctional detection instrument. Pictures were taken at an interval of 6 h to calculate the number of cells and the relative cell proliferation rate.
Relative cell proliferation rate = cell proliferation number in the drug group/ cell proliferation number in the control group × 100%
2.5. MDC staining
Take the logarithmic growth phase cells, make a single cell suspension and inoculate it overnight in a 6-well culture plate. After 24 h of drug intervention, the culture was terminated, the supernatant was discarded and washed with phosphate buffer solution(PBS), 1 mL of 50 μM MDC solution was added to each well, incubated for about 40 min, washed with PBS three times, and photographed under a CKX31 inverted fluorescence microscope. Image-Pro Plus 6.0 software was used to calculate the fluorescence intensity.
2.6. Transmission Electron Microscope
The cells were inoculated in culture flasks at 5 × 105 and cultured overnight. After drug intervention for 24 h, the cells were collected into Ep pendorf (EP) tubes, and centrifuged at 1000g for 5 min, the supernatant were discarded, and the cells were resuspended by adding 1 mL PBS. The centrifugation was repeated twice, the supernatant discarded, 1 mL 2.5% glutaraldehyde (prepared with PBS) was added to each tube, before placing in a refrigerator at 4 ℃ for more than 4 h, and then fixed with 1% osmium acid fixative for 2 h. After dehydration with gradient acetone, soaking, embedding, curing, and ultrathin sectioning, it was stained with 3% uranyl acetate and lead nitrate, and the film was observed and photographed with a Hitachi H7700 transmission electron microscope.
2.7. Western Blot analysis
After 24 h of drug intervention, the Petri dish was placed on ice to extract the total protein. Protein extracts were quantified using a BCA protein assay kit (Lianke, Hangzhou, China). Equal amounts of proteins were loaded on 12% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% fat-free milk in TBST at room temperature for 1 h, then incubated with primary antibodies anti-β-actin (1:1000, 3700S), anti-LC3 a/b (1:1000, 12741P), anti-Beclin1 (1:1000, 3495P), anti-P62 (1:1000, 5114S), anti-proliferating nuclear antigen (PCNA, 1:1000, 13110 ) , anti-p-c-Raf (1:1000, 9427), anti-p-MEK (1:1000, 9154), and anti-p-ERK1/2 (1:1000, 4370S) at 4 °C overnight (all from Cell Signaling Technology, Beverly, MA, USA). After primary antibody incubation, membranes were washed three times in TBST then incubated with secondary antibody (1:5000, LI-COR, Lincoln, NE, USA) for 1 h at room temperature. After washing, signals were visualised by an Odyssey Infrared Imaging System (Lincoln, NE, USA). Endogenous beta-actin was used for normalisation.
2.8. Statistical analysis
SPSS 23.0 software (IBM Corp., Armonk, NY, USA) was used to analyze the data results. All experiments were performed at least three times. Results are presented as the mean ± standard deviation (SD). The single-factor design and the measurement data comparison between multiple groups use single-factor analysis of variance. Multiple comparisons that meet the homogeneity of variance use the LSD test. The Dunnet T3 test was used for multiple comparisons that met the homogeneity of variance,the rank sum test was used for those that did not meet the normal distribution. If P < 0.05, the difference was considered to be statistically significant. All analysis diagrams were made using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA).