Antibodies and reagents
LiCl was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against caspase8 (cat no. 9746), caspase9 (cat no. 9502), Bip (cat no. 3183), poly (ADP-ribose) polymerase (PARP; cat no. 9542), phospho-eIF2α (Ser51) (D9G8) (cat no. 3398S), eIF2α (cat no. 9722S), IRE1α (cat no. 3294), ATF-4 (D4B8) (cat no. 11815S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting caspase3 (cat no. NB100-56708) were purchased from Imgenex (Novus Biologicals, LLC, Littleton, CO, USA). The NOXA antibody (cat no. OP180) was obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). Antibodies against CHOP (cat no. sc-7351) and Mcl-1 (cat no. sc-12756) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The GSK3β antibody was obtained from Abcam (cat no. ab32391; Abcam; UK).
Cell lines and cell culture
The human choroidal melanoma lines OCM1 and M619 were obtained from the China Centre for Type Culture Collection (Wuhan, China) and were grown in monolayer cultures at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. OCM1 cells were cultured in Dulbecco’s modified Eagle’s medium containing 5% foetal bovine serum, and M619 cells were cultured in RPMI-1640 medium containing 5% foetal bovine serum (both Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA).
Cell viability assay
Cells were seeded in 96-well plates at a density of 5.0×103 cells/well and were then treated with the indicated concentrations of LiCl on the second day. The cells were cultured with chemotherapeutics for 24, 36 or 48 h then subjected to the MTT assay. Each sample was incubated with 20 µl of (5 mg/ml) MTT (Sigma‑Aldrich; Merck KGaA) at 37°C for 4 h. Then, the solution was discarded, and 100 μl of dimethyl sulfoxide was added. The absorbance at 495 nm due to formazan was measured by an ELISA Multiskan reader (Thermo Fisher Scientific, Inc.).
Colony formation assay
The cells were seeded into 6-well plates at a density of 1× 104 cells per well. After the cells were incubated overnight, the cells were treated with 0, 2.5, 5, or 10 mM LiCl and incubated for approximately 2 weeks. During this period, the indicated concentrations of LiCl were added to the wells every 72 h. When the cell colonies were visible to the naked eye, the cells were subjected to the colony formation assay. The culture solution was discarded, and the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20min. After that, the cells were washed with PBS 3 times, stained with 1% crystal violet for 20min, washed out slowly with water, and dried in the air. The number of cell colonies (>50 cells) were counted under a microscope.
Apoptosis was evaluated according to a previously described protocol. The Annexin V-FITC/ propidium iodide (PI) apoptosis detection kit was purchased from BIO-BOX Biotech (Nanjing, China). The cells were treated with various concentrations of LiCl for 36 h, and then 2×106 cells were collected, washed with prechilled PBS and resuspended in 500 μl of binding buffer. Then, each sample was incubated with 5 μl of Annexin V-FITC and 5 μl of PI for 15 min in the dark at room temperature. Then, the cells were analysed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed using FlowJo software (version 7.2.2; Tree Star, Inc. San Carlos, CA, USA).
Western blotting analysis
Whole-cell protein lysates were prepared and analysed by western blotting according to a previously described protocol. After being harvested and rinsed with prechilled PBS, the cells were lysed, and the extract was centrifuged at 12,000 x g at 4°C for 15 min. Whole‑cell protein lysates (40 µg) were electrophoresed on 12% denaturing polyacrylamide slab gels and transferred to Hybond-enhanced chemiluminescence (ECL) membranes through electroblotting. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with specific primary antibodies and subsequently with secondary antibodies. Antibody binding was detected using an ECL system (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The protein expression levels were quantified using ImageJ software (version 1.6.0_24; National Institute of Health, Bethesda, MD, USA).
Plasmid transient transfection
The pcDNA3.1-Mcl-1 plasmid was obtained from Addgene (Cambridge, MA, USA). OCM1 and M619 cells were seeded in 6-well plates and transfected with pcDNA3.1 and pcDNA3.1-Mcl-1 plasmids using X-treme GENE HP DNA Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol. Then, the cells were treated with the indicated concentration of LiCl for 24 h and subjected to western blotting and apoptosis analysis.
Transfection with siRNA
Previously described siRNAs targeting sequences of CHOP and GSK3β were synthesized[15, 16]. Transfection with siRNA was conducted using jetPRIME Transfection Reagent (Polyplus Transfection SA, Illkirch, France) following the manufacturer’s protocol. Choroidal melanoma cells were seeded in 6-well plates and transfected with control or target siRNA on the second day. Two days later, the cells were treated with various concentration of LiCl for another 24 hours. Then the cells were harvested for western blotting analysis.
In vivo tumorigenesis analysis
Five-week-old BALB/c nude male mice were obtained from Beijing Vital River Laboratory Animal Technology (Co., Ltd./Charles River Laboratories, Beijing, China). The BALB/c nude male mice were randomly divided into a normal saline group and a LiCl group with 5 mice per group. M619 cells were subcutaneously injected into the right flank region of each mouse (3 × 106 cells in 100 μl of PBS). The tumour size was recorded every 3 days beginning on the day the tumours were first visible. Tumour volume was calculated using the following equation: Volume=(width2×lenghth)/2. The LiCl group was treated with LiCl (141.3 mg/kg; i.p., daily) for 2 weeks, while the normal saline group received an equal volume of normal saline. Finally, the mice were sacrificed on the 15th day, and the tumour tissues were collected for western blotting and immunohistochemical analysis. The study was approved by the Shandong University Second Hospital Ethics Committee.
Immunohistochemical (IHC) analysis was performed according to a previously described protocol. The tumour tissues were fixed in 10% formalin. Following proper dehydration, the tumours were embedded in paraffin and then cut into 5-μm-thick sections. After deparaffinization and rehydration, the sections were submerged in sodium citrate antigen retrieval solution (pH 6.0) and microwaved for 8‑15 min for antigen retrieval. Endogenous peroxidase was deactivated by H2O2. Then, the slides were blocked using 10% goat serum and incubated in the corresponding primary antibodies overnight at 4°C. After being washed, the sections were incubated with HRP-conjugated secondary antibody for 50 min at room temperature, followed by incubation with 3,3-diaminobenzidine (DAB) solution and counterstaining with haematoxylin. The anti-Ki67 rabbit mAb (cat no. GB 13030-2) was purchased from Wuhan Servicebio Technology Co., Ltd. (China).
All experiments were repeated at least three times. All statistical analyses were performed using SPSS statistical software (version 20.0; IBM Corp., Armonk, NY, USA). The data are represented as the mean ± S.D. of at least three independent assays performed in duplicate or triplicate. An unpaired t-test was used to compare differences between two groups, and one-way ANOVA was used to compare differences among more than two groups. A value of P < 0.05 was considered statistically significant.