Reagents and antibodies
The MCL1 (16225-1-AP), HA(51064-2-AP), Alpha Tubulin (11224-1-AP) and FBXW7 (28424-1-AP) antibodies were purchased from Proteintech Group (USA), and antibodies CDK1( #77055), CDK2 (#78B2), Cleaved Caspase 3 (#14220), Cleaved Caspase 9 (#52873), Cleaved PARP (#5625), PARP (#9542), Bim (#2933), BAX (#5023) BCL2 (#15071) were purchased from Cell Signaling Technology (USA). 5-Bromo-2-deoxyUridine (BrdU) (ab8152) and Ki67 (ab15580), β-TrCP (ab71753), HUWEI/mule (ab70161) antibodies were purchased from Abcam (USA). The One Step TUNEL Apoptosis Assay Kit (C1089) was purchased from Beyotime (China). Cycloheximide (C7698), 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT; M5655), DMSO (D5879), and MG132 (M7449) were purchased from Sigma-Aldrich (USA). Lycorine hydrochloride was purchased from MUST BIO-TECHNOLOGY (Cheng Du, China). Alexa Fluor 488 goat anti-rabbit IgG (H+L) (35552) was purchased from Invritrogen (USA). The puromycin (A1113803) was purchased from Life Technologies (New York, USA). The transfection reagent Lipofectamine™ 2000 was obtained from Thermo Fisher Scientific (New York, USA). HRP goat anti-mouse (Cat. No: 5220-0341) and goat anti-rabbit antibodies (Cat. No: 5220-0336) were purchased from Seracare company (USA). Annexin V-APC (2005128) and propidium iodide (2048964) were obtained from Invitrogen (USA). The propidium iodide (2031727) for cell cycle was purchased from Invitrogen (USA).
Cell Culture
MKN-45, SGC-7901 and 293-FT cell lines were purchased from American Type Culture Collection (ATCC, USA). MKN-45-R and SGC-7901-R cell lines were obtained from our laboratory. All cells were cultured in 1640 medium (with 10% fetal bovine serum, 1% Penicillin-Streptomycin Solution) at 37 °C in a humidified atmosphere containing 5% CO2.
Cell viability (MTT), BrdU staining assays, western blotting analysis and qRT-PCR
Cell viability (MTT), BrdU staining assays, western blotting assays and qRT-PCR assays were analyzed as previously described [30].
Flow cytometry
(a) Cell cycle analysis: MKN-45 and SGC-7901 cells were harvested after added with LH for 48h, and fixed in 70% ethanol at 4°C for 24h. Washed with PBS, the cells were added into 50 µg/mL RNase A and 1% propidium iodide for 35 minutes at 37 ℃. Subsequently, the BD Accuri C6 flow cytometer and FlowJo software were used to analyze the change of DNA.
(b) Cell apoptosis analysis: MKN-45 and SGC-7901 cells were harvested after being treated with LH for 48h. After being washed with 1x Bingding buffer, the cells were incubated with Annexin V-APC (0.5%) and propidium iodide (1%) at room temperature for 20 min. Subsequently, the BD Accuri C6 flow cytometer and FlowJo software were used to analyze the change of apoptosis following the manufacturer’s instructions.
TUNEL experimental analysis
Twenty thousand cells were evenly spread on the 24-well cell culture plate. After 24 hours, LH (20 μm) and DMSO were added respectively, incubated for 48 hours. Washed it with PBS once. Then 4% PFA fixed the cells for 30 minutes. Washed again with PBS. Added PBS containing 0.5 % Triton X-100 and incubated at room temperature for 5 minutes. Cleaned it with PBS twice. Added the prepared TUNEL test solution (50 μL) every well, incubated at 37 ℃ in the dark for 60 minutes. Then washed three times with PBS. It was observed by fluorescence microscope after sealing with anti-fluorescence quenching solution. The excitation wavelength of Cy3 is 550 nm, and the emission wavelength is 570 nm (red fluorescence).
Transfection and infection
The interference plasmid shMCL1 (#1; #2) and the negative control (SHC002) were purchased from Sigma-Aldrich (USA). The MCL1-overexpression plasmid (pCDH-CMV-MCS-EF1-copGFP-MCL1) was purchased from YouBio (Changsha, China) [19]. Transfection and infection were carried out as manufacturer’s instructions. Firstly, the liposomes and the packaging plasmids (PLP1, PLP2, VSVG, target plasmids: shMCL1 or OE-MCL1) were transferred into 293-FT cells. The virus was harvested 48 hours later. Gastric cancer cell lines MKN-45, SGC-7901 were infected with the harvested virus.
Subcutaneous tumour xenografts (CDX model)
Experiments in vivo were carried out with the approval of the Committee for Animal Protection and Utilization of Southwest University. According to the Guidelines for Animal Health and Use (Ministry of Science and Technology, China, 2006), all experiments were conducted orderly. Purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China), six five-week-old female nude mice were raised and observed in SPF room for a week to adapt to the new environment. On June 20, 2019, SGC-7901 cells (1 × 106 cells per mouse) suspended in 0.1 ml serum-free RPMI-1640 were subcutaneously inoculated on the left and right upper back of mice. In order to alleviate the pain of mice, we used the isoflurane for nasal anaesthesia before subcutaneous injections. Isoflurane can make the mice enter anesthesia state faster and recover quickly. After the anesthesia stops, the mice commonly wake up within 2 minutes, and the control of anesthesia depth was very easy. If the mice were found to be out of condition during the operation, the anaesthesia machine would be shut off immediately, and the mice would be rescued quickly. Hence, the safety of mice was guaranteed. Isoflurane can be completely discharged from the alveoli through respiration without affecting metabolism in vivo, and has no effect on the experimental results. In addition, isoflurane is widely used in animal experiments in the world. The mouse anaesthesia system was purchased from Reyward Life Technology Co., Ltd. (Shenzhen, China). All experiments were performed on a sterile workbench of an SPF room [31]. Before and after subcutaneous injection, 75% medical alcohol was used to disinfect the epidermis of mice. After one week injection, they were randomly divided into two groups, which were respectively treated with DMSO and LH (30mg / kg) once a day for 16 days. During this period, tumor volume [tumor volume= (length × width 2)/2] was measured every two days under strict and standardized feeding conditions. Before tumor collection, nasal anesthesia (isoflurane) was used in mice to relieve pain. Then, the mice were killed by cervical dislocation. The tumor was removed, and the weight was recorded. The bodies were frozen at -20°C before transferring to Laibite Biotech Inc. (Chongqing, China) for incineration. Finally, the tumor was photographed and recorded, which will be used for subsequent immunohistochemistry experiments.
PDX experiment
Purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China), nine five-week-old female nude mice were raised and observed in SPF room for a week to adapt to the new environment. On November 10, 2019, with the consent of the patient's family, the GAM-AD tumor mass from The Ninth People’s Hospital of Chongqing was cut into even 2x2x2mm pieces and suspended in 0.1ml serum-free RPMI-1640, and planted into subcutaneous tissue of mice in equal volume. The whole process is consistent with the previous subcutaneous tumour xenografts experiment. Two weeks after operation, they were randomly divided into three groups, respectively treated with DMSO, LH (30mg/kg) and LH (30mg/kg)+HA14-1 (2.5mg/kg) once a day for 16 days. In addition, after one week operation, the weight of the mice was recorded every four days. The process of tumor collection was also consistent with the aforementioned subcutaneous tumour xenografts experiment. The tumor weight was recorded, and the tumor was photographed and recorded, which will be used for subsequent immunohistochemistry experiments.
Immunoprecipitation (Co-IP)
Protein A/G Magnetic Beads (HY-K0202) were purchased from MCE (Monmouth Junction, NJ, USA). Cells treated with LH or DMSO were lysed and collected. Then, operate according to the instructions.
Ubiquitination assay
Firstly, the HA-Ub plasmid was transiently transferred into 293-FT. Then 293-FT cells were harvested before incubating with proteasome inhibitor MG132 (50 μg/ml) from Selleck (Houston, USA) for 6 h. The harvested cells were lysed by the IP lysis solution. Subsequently, incubated with anti-MCL1 (1%) or IgG at 4 ℃ for overnight. The second day, the Protein A/G Magnetic Beads were added following with the instruction. Then, the proteins adsorbed from magnetic beads were analyzed by western blotting. HA tag antibody (51064-2-AP) from Proteintech Group (USA) was used to check the interaction between MCL1 and Ub.
Immunohistochemistry Assay
After the tumor tissues were paraffin sectioned, they were incubated with MCL1 (1:100) FBXW7 (1:100) or Ki67 (1:100) antibodies at 4°C for overnight. Then the paraffin sectioned were incubated with HRP-conjugated secondary antibodies for 3 hours at room temperature. Subsequently, they were stained by DAB, and tissues were counterstained with hematoxylin. Finally, photographs were taken by the inverted microscope.
The screen of BCL2-drug-resistant-cell-lines
The MKN-45 and SGC-7901 were cultured in 1640 medium with HA14-1(9 μM) for a week. Then the dead cells were washed with PBS. The remaining living cells were diluted in 96-well plate by gradient dilution method and maintained a high concentration of HA14-1. Continue to cultivate for 3 weeks. Finally, the surviving monoclonal cells were selected, and amplification cultured. Finally, the HA14-1-resistant cell lines (MKN-45-R and SGC-901-R) were obtained.
Autophagy flux detection
The mRFP-GFP-LC3-adenovirus (HB-AP2100001) was purchased from HanBio (Shanghai, China). Subsequently,mRFP-GFP-LC3B-adenovirus was added into the medium of MKN-45 and SGC-7901, cultured for 24h. Then, removed the old medium and added the fresh 1640 medium (with 10 % fetal bovine serum, 1% Penicillin, Streptomycin, and 20 μM LH). After 48h, confocal microscopy was used to record the experimental results.
Statistical analysis
Statistical analyses were obtained by the Graphpad prism software. Data were showed as mean ± SEM and analyzed by unpaired 2-tailed t-tests. P-values of <0.05 (*), <0.01 (**) and <0.001 (***) were considered statistically significant.