Drugs and chemicals
BON was kindly supplied by Professor Yun-ying Xie at our institute. Other chemicals were purchased from Selleck Chemicals (Houston, TX, USA) or Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
Cell lines and cell culture
All cell lines were acquired from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 (NCI-H460 cells), F12 (A549 cells), MEM (HT-1080 cells) or DMEM (MDA-MB-231 cells) medium (HyClone, UT, USA), and that was supplemented with 10% fetal bovine serum (Gibco, NY, USA) was supplemented. All cells were cultured in a humidified atmosphere at 37˚C with 5% CO2.
Western blot assay
Western blot assay has been described in detail previously . Antibodies against PD-L1 (#13684), PARP-1 (#9542), cleaved caspase-3 (#9661), p-AMPKα (#2535), AMPKα (#2532), LC3B (#2775) and HRD1 (#14773) were purchased from Cell Signaling Technology (Boston, MA, USA). The β-actin (sc-47778) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody dilution ratios for β-actin and cleaved caspase-3 were 1:100,000 and 1:500, and those for the others were all 1:1000.
The Cell Counting kit-8 (CCK-8) assay was performed according to the method published in our previous paper . CCK-8 was obtained from Bimake (Houston, TX, USA). The operation steps and calculation formula for cell survival rates have been described previously.
Mice xenografts and drug administration
Female BALB/c nude mice weighing 18-22 g were purchased from SiPeiFu Beijing Biotechnology Co., Ltd (Beijing, China). NCI-H460 cells were used for the establishment of a xenograft model. Tumor-bearing mice were randomly divided into three groups that included the vehicle group, BON 1 mg/kg group, BON 9 mg/kg group. The mice were administrated with BON (i.p.) every two days. The body weights and tumor volumes were measured. After seven drug administrations, the mice were sacrificed and the tumor masses were separated. The care and sacrifice of nude mice were performed according to the protocols approved by the Animal Care and Use Committee of the Institute of Medicinal Biotechnology.
Detection of apoptotic cells by Annexin V/PI staining
The collected cells were stained with annexin V and PI according to the manufacturer’s the instructions. The fluorescence intensities were measured using a flow cytometer (BD FACSCalibur, BD Biosciences). CellQuest Pro software version 5.1 was used for data analysis.
The siRNAs targeting PD-L1, AMPKα1, AMPKα2 and HRD1 were synthesized by Guangzhou RiboBio Co., Ltd (Guangzhou, China). The detailed transfection steps and conditions have been described previously . The sequences of specific siRNAs are as follows:
AMPKα1#2: 5’- CCAGGATCCTTTGGCAGTT-3’;
HRD1#2: 5’- AAACGGTGAAGGCCAGACA -3’.
Data from three separate experiments are shown as mean ± SD. Statistical analyses were performed using SPSS software version 19.0. Comparisons between two groups were conducted using Student’s t-test, whereas the differences among more than two groups were compared by one way ANOVA. Statistical significance was indicated as * P<0.05, ** P<0.01 and *** P< 0.001.