Cell culture and treatment
The human breast cancer (BC) cell lines (MDA-MB-231 and MCF-7) were purchased from American Type Culture Collection (ATCC, USA), and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, USA) containing 10 % fetal bovine serum (FBS, Hyclone, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in an incubator with 5 % CO2 humidified atmosphere at 37 ℃. The cells were harvested for further experiments until the cell confluency reached about 70-80 %, and were subjected to increasing concentrations (0.01-1 μg/ml) of doxorubicin (Sigma, USA) in a step-wise manner over a period of 6 months to generate doxorubicin-resistant BC (DR-BC) cells based on the experimental protocol provided by the previous publication [32, 33]. To sustain the doxorubicin-resistant phenotypes, the DR-BC cells were cultured in the DMEM medium supplemented with 0.01 μg/ml doxorubicin. After that, the DR-BC cells were pre-treated with 10 μmol/L of AMPK inhibitor compound C, 6 μmol/L ULK1 inhibitor SBI-0206965 and 12mmol/L of autophagy inhibitor 3-methyladenine (3-MA) for 24 h, and subsequently stimulated with high-dose doxorubicin (20 μg/ml) for 0 h, 24 h, 48 h and 72 h.
Western Blot analysis
The total protein was extracted from the BC cells by using a commercial RIPA lysis buffer, and the following Western Blot analysis was conducted to examine the protein levels of the involved genes, and all the detailed experimental procedures had been well documented in the previous work [32, 33]. The primary antibodies were listed as follows: Cyclin D1 (1:1500, Abcam, UK), CDK2 (1:2000, Abcam, UK), cleaved caspase-3 (1:2000, Abcam, UK), Bax (1:2000, Abcam, UK), β-actin (1:3000, Abcam, UK), LC3B (1:1000, Abcam, UK) and p62 (1:1000, Abcam, UK).
Cell counting kit-8 (CCK-8) assay
The BC cells were subjected to different treatments, and cell proliferation was evaluated by using the commercial CCK-8 assay kit purchased from Abmole (USA) according to the manufacturer’s protocol. Briefly, the cells were seeded into the 96-well plates, and cultured in the incubator for 0 h, 24 h, 48 h and 72 h, respectively. Next, the cells were incubated with 20 μl CCK-8 reaction solution, and the optical density values were examined to reflect relative cell proliferation abilities.
Colony formation assay
The BC cells were harvested and administered with different stimulation, after that, the cells were cultured in the 6-well plates at the concentration of 500 cells per well. After 14 days culture in the incubator, the cells were stained with 0.4 % crystal violet for visualization. Next, the cells were observed and photographed under light microscope, and the cell colonies containing at least 10 cells were counted.
Trypan blue staining assay
The BC cells were collected, and cell viability was determined by using the trypan blue staining method. The cells were stained with the trypan blue staining solution for 20 min at 37 ℃, and the dead blue cells were observed, photographed and counted under light microscope. The formula for calculating cell viability was shown as follows: cell viability (%) = (total cell numbers – dead blue cell numbers)/total cell numbers * 100%.
Measurement of cell apoptosis
The commercial apoptosis detection kit (BD Bioscience, USA) was purchased to measure cell apoptosis ratio in BC cells with differential stimulation, and the detailed experimental procedures were recorded in the manufacturer’s instruction. In brief, the cells were double stained with Annexin V-FITC and PI for 30 min at room temperature without light exposure, and a flow cytometer (ThermoFisher Scientific, USA) was used to examine and count the numbers for apoptotic cells.
Observation of autophagosomes by electronic microscope (EM)
The BC cells, including DS-BC and DR-BC cells, were fixed with 0.1 M sodium cacodylate-buffered 2 % (wt/vol) glutaraldehyde at 4 ℃ overnight, and pelleted in agarose, rinsed in distalled water and stored in 70 % ethanol. After that, the cells were dehydrated and stained with 2 % aqueous uranyl acetate for 5 min, and Reynolds lead citrate for 3 min at room temperature. Finally, the cells were observed and photographed by an electronic microscope (EM) at 80 kV.
All the data were presented as Means ± Standard Deviation (SD), and the data were analyzed and visualized by suing the SPSS 18.0 software and GraphPad Prism 7.0 software. Specifically, the comparisons of means from two groups were conducted by using the unpaired Student’s t-test, and the means from multiple groups were compared by using the one-way ANOVA analysis. P < 0.05 was regarded as statistical significance, and marked by “*” in the Figures. Each experiment repeated at least 3 times.