Ki 67

MTT, crystal violet solution, PI, RNAse A and DAPI were purchased from Beijing Solarbio Science & Technology Co., Ltd. Western blotting reagents were obtained from Beyotime Institute of Biotechnology. An Annexin V/PI apoptosis detection kit was purchased from 7 Sea Biotech (http://www.7seapharmtech.com/).
Primary antibodies against the following proteins were purchased from Cell Signaling Technology, Inc. (CST): AKT (cat. no. 9272), phosphorylated (p)-AKT (Ser473; cat. no. 4060), mTOR (cat. no. 2983), p-mTOR (Ser2448; cat. no. 5536), p70S6K (cat. no. 9202), p-p70S6 kinase (p70S6K; Ser371; cat. no. 9208), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1; cat. no. 9644), p-4E-BP1 (Thr37/46; cat. no. 2855), Beclin-1 (cat. no. 3495), p-Beclin-1 (Ser93; cat. no. 14717), Cyclin D1 (cat. no. 2978), Cyclin E1 (cat. no. 4129), cleaved caspase-3 (cat. no. 9664), autophagy related 5 (ATG5; cat. no. 9980) and GAPDH (cat. no. 5174). LC3-I/II (cat. no. ABC929) was purchased from Sigma-Aldrich (Merck KGaA) and ki67 (cat. no. ab15580) was obtained from Abcam. Secondary horseradish peroxidase-linked antibodies against rabbit (cat. no. 7074) and mouse (cat. no. 7076) were purchased from CST. Small interfering RNA (si)ATG5 was purchased from Shanghai GenePharma Co., Ltd., and Lipofectamine^® 2000 was obtained from Thermo Fisher Scientific, Inc.

Mouse subcutaneous tumors were collected following euthanasia. Subsequently, tissue sections (4-µm) were dewaxed with xylene (twice) for 10 min and hydrated using a descending alcohol series (100% twice, 90% twice, 75% twice) for 3 min at room temperature. Antigens were retrieved in a 95°C water bath for 20 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 20 min at room temperature. After blocking with 5% BSA (Sigma-Aldrich; Merck KGaA), the sections were incubated with primary antibody (Ki-67, 1:200; cat. no. 9027S; Cell Signaling Technology, Inc.) at 4°C overnight. The next day, sections were incubated with secondary antibody (1:200; cat. no. ZDR-5306; OriGene Technologies Inc.) for 2 h at 37°C, followed treating with DAB Chromogen (1 drop of the DAB Chromogen per ml of substrate buffer; Dako; Agilent Technologies, Inc.) and hematoxylin (cat. no. C0107; Beyotime Institute of Biotechnology) for 2 min at room temperature. Finally, the slides were dehydrated and fixed by resin and observed with an Olympus inverted microscope (Olympus Corporation; magnification, ×200).

To analyze the effects of betulin (3) on various protein markers, 20,000 NCI-H460 cells were seeded in a 24-well plate with or without betulin. After 48 hours treatment, media was discarded and cells were carefully and thoroughly washed with PBS. Then cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Again, wells were washed with PBS and 150 µL Triton X-100 was added to the wells for 10 minutes. Cells were incubated with blocking solution for 30 minutes in a humidified environment followed by addition of primary antibody (1:100 dilution in blocking solution) overnight at 4°C. The next day, cells were washed with PBS and respective secondary antibody (Thermo Fisher Scientific) was added to the wells for 1 hour. Finally, DAPI staining was done followed by observing expression of markers under fluorescent microscope at 10× magnification. The primary antibodies used against the markers include BCL-2L1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), Ki-67 (EMD Millipore, Billerica, MA, USA), caspase-3 (EMD Millipore), caspase-6 (EMD Millipore), caspase-8 (EMD Millipore), and osteopontin (Abcam, Cambridge, MA, USA).