Cell lines, cell culture, and transfection of lentiviral vectors
The human HCC cell lines HuH7, PLC/PRF/5, MHCC97H, Hep3B, and HepG2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and the cell library of the Chinese Academy of Sciences. All cell lines were cultured in the providers’ recommended medium supplemented with 10% fetal bovine serum (Gibco, South America origin), 100 μg/mL penicillin (Yeasen, Shanghai, China), and 100 μg/mL streptomycin (Yeasen, Shanghai, China) at 37 °C in a humidified 5% CO2-containing atmosphere. A KSR2 lentiviral vector was constructed (Genomeditech, Shanghai, China). Stable transfectants were characterized by quantitative real-time polymerase chain reaction (qPCR) or western blotting. Table S2 lists the targets of shKSR2.
RNA extraction and real-time polymerase chain reaction assay
Total RNA was extracted from HCC cells and tissues using TRIzol Reagent (Sigma-Aldrich, USA) according to the manufacturer’s protocol. cDNA was synthesized by random primers and the HifairII 1st strand cDNA synthesis kit (Yeasen, Shanghai, China). Real-time polymerase chain reaction (qPCR) was performed using the Hieff qPCR SYBR green master mix (Yeasen, Shanghai, China). PCR conditions were: 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Table S3 lists the PCR primers. ACTB was used as the internal control.
Protein extraction and western blotting
Cell and tissue proteins were extracted using lysis buffer for WB/IP assays (Beyotime Institute of Biotechnology, China) with a phosphorylated protease inhibitor cocktail (Yeasen, Shanghai, China) and a proteinase inhibitor cocktail (Yeasen, Shanghai, China). Protein concentrations were determined using a BCA protein quantification kit (Yeasen, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were incubated with primary antibodies overnight at 4 °C and probed with secondary antibodies at room temperature for 1–2 h. The following antibodies were used: anti-KSR2 (1:500, Abnova, Taiwan, China), anti-CRAF (1:1000, CST, Massachusetts, USA), anti-p-CRAF (1:1000, CST, Massachusetts, USA), anti-MEK1/2 (1:1000, CST, Massachusetts, USA), anti-p-MEK1/2 (1:1000, CST, Massachusetts, USA), anti-ERK1/2 (1:1000, CST, Massachusetts, USA), anti-p-ERK1/2 (1:1000, CST, Massachusetts, USA), anti-14–3-3ζ (1:1000, CST, Massachusetts, USA), anti-Cleaved-Caspase 3 (1:500, Arigo, Taiwan, China), and anti-β-actin (1:1000, CST, Massachusetts, USA).
Cell transfections
Small interfering RNA (siRNA) oligonucleotides for 14–3-3ζ were designed and synthesized by Genomeditech (Shanghai, China). Table S4 shows the primer sequences for the siRNAs. Transient transfection was performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. After transfection for 48 h, cells were used for functional assays, including cell viability assays, RNA extraction, and western blotting.
Colony formation assays
For cell viability assays, 1000 cells were placed in a 6-well plate and maintained with 10% FBS-Dulbecco’s Modified Eagle’s Medium (DMEM) for 2 weeks. Colonies were fixed with methanol and stained with 0.1% crystal violet for 10 min. A clone including more than 50 cells was counted as positive.
Wound healing migration
Cells were seeded into 6-well plates and grown to approximately 90% confluence. Cell monolayers were scratched with a 20-μL sterile pipette tip. Cells were rinsed with phosphate-buffered saline and cultured in DMEM supplemented with 1% fetal bovine serum. Cell migration was photographed 0 h and 48 h after scratching using an inverted microscope (Olympus, Tokyo, Japan).
Invasion assays
Matrigel invasion assays were performed in 24-well plates with 8 μm-pore size chamber inserts (Corning, New York, USA). Upper chambers were precoated with 60 μL of Matrigel. After 30 min coating with Matrigel, 2 × 104 cells in 200 μL of serum-free culture medium were seeded into each well of the upper chamber, and 500 μL of medium supplemented with 10% fetal bovine serum (FBS) were added to the lower chamber. Chambers were incubated at 37 °C with 5% CO2 for 72 h, and then cells that migrated through the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 30 min, and imaged and counted under a light microscope (Olympus, Tokyo, Japan).
In vivo tumor growth
All animal experiments were approved by the Animal Ethics Committee of Zhongshan Hospital affiliated with Fudan University. Experiments used 6-week-old male BALB/c-nu/nu mice raised by Zhongshan Hospital. Mice were housed and experiments performed in a specific pathogen-free (SPF) animal facility. For the in vivo xenograft assays, 5 × 106 PLC/PRF/5 cells stably expressing shKSR2 or the negative control and 5 × 106 PLC/PRF/5 cells stably expressing KSR2 or the lentiviral vector were individually subcutaneously inoculated into the dorsal right flanks of nude mice (6 per group). Size of the tumors was measured twice a week. Tumor volume was measured using calipers and calculated as V = (length × width2)/2. After 3–4 weeks, mice were euthanized and tumors were harvested and fixed in 10% neutral phosphate-buffered formalin. Fixed tumors were stained via immunohistochemistry. In another experiment, BALB/c-nu/nu mice were injected subcutaneously with PLC/PRF/5 cells (5 × 106 cells/mouse) and treated with sorafenib (20 mg/kg gavage, once every other day) at day five for two weeks. On day 12 after the start of treatment, tumors were removed.
Co-immunoprecipitation (co-IP) and protein mass spectrometry
Cells were cultured to > 90% confluence then lysed in immunoprecipitation lysis buffer (Beyotime Shanghai, China) with protease and protein phosphatase inhibitors. Lysates were incubated with 20 μL of protein A/G magnetic beads (MedChemExpress, New Jersey, USA) for 2 h. After incubating, the beads were removed, and 5–10 μL of primary antibody (KSR2) or isotype IgG was added to the supernatant and the samples mixed with gentle rocking at 4 °C overnight to capture the fusion proteins. Then, 20 μL of protein A/G beads was added and the immunoprecipitation mixes incubated for 2 h. Magnetic beads were collected by placing the tube in the appropriate magnetic separator. Beads were washed three times with cooled PBS buffer to remove nonspecifically bound proteins. Bound fusion proteins were eluted from the beads. Peptides were then analyzed (Bioprofile, Shanghai, China) with the Easy-nLC1200 chromatographic system (Thermo Fisher, Massachusetts, USA) and Q-exactive Plus mass spectrometer (Thermo Fisher, Massachusetts, USA). Protein identification was performed using MaxQuant1.6.1.0.
Immunofluorescence (IF)
HCC tissue was fixed in 4% paraformaldehyde at 4 °C and then embedded in optimal cutting temperature compound and sectioned (20-μm thick) using a freezing microtome (Leica, Witzlar, Germany). After antigen retrieval, sections were blocked in phosphate-buffered saline supplemented with 5% bovine serum albumin for 1 h. Sections were then incubated with primary antibodies overnight at 4 °C. Secondary antibodies were Alexa Fluor 594-conjugated anti-rabbit IgG (1:200, Yeasen, Shanghai, China) or Alexa Fluor 488 anti-mouse IgG (1:200, Yeasen, Shanghai, China). Slides were exposed to DAPI nuclear stain (1:200, Yeasen, Shanghai, China) for 20 min, before being sealed with antifade mounting medium (Yeasen, Shanghai, China) and glass coverslips. Images were captured using a fluorescence microscope (Leica, Witzlar, Germany).
Clinical specimens and tissue microarray (TMA)
A tissue microarray containing 198 paired HCC tissues and matched adjacent noncancerous tissues as well as fresh human hepatocellular carcinoma tissues and matched adjacent noncancerous tissues for qPCR and western blot analyses were collected from the Department of Liver Cancer at Zhongshan Hospital affiliated with Fudan University, Shanghai, China, from 2007 to 2020. All specimens were immediately snap-frozen in liquid nitrogen after surgical removal. Informed consent was obtained from all subjects to use the specimens described in this study. The procedure related to human subjects was approved by the Ethics Committee of the Fudan University.
Immunohistochemistry (IHC)
Tissues were fixed in 4% paraformaldehyde, washed three times with PBS, transferred to 70% ethanol, and then embedded in paraffin and sectioned according to standard procedures. Sections were dewaxed with a graded ethanol series. After antigen retrieval, the tissues were stained using the Streptavidin Peroxidase IHC assay kit (ZSGB-Bio, Beijing, China). The primary antibodies were anti-KSR2 (1:100, Abnova, Taiwan, China), anti-14–3-3ζ (1:800, CST, Massachusetts, USA), anti-ERK1/2 (1:400, CST, Massachusetts, USA, anti-Cleaved-Caspase 3 (1:100, CST, Massachusetts, USA) and anti-KI67 (1:100, Abcam, Cambridge, UK). The average gray value of each image was used to quantify the expression level using Image-Pro Plus 6.0 software.
Apoptosis analysis
Cell apoptosis was analyzed using the Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit (Bio-platform, Shanghai, China) according to the manufacturer’s instructions. PLC/PRF/5 cells were stained with FITC and PI and then analyzed by fluorescence-activated cell sorting using the LSRFortessa (BD Biosciences, CA, USA). Cell apoptosis data were analyzed by the Flowjo V10.2 software.
Statistical analysis
The statistical differences between groups were analyzed by χ2 analysis or two-tailed Student’s t tests. The Kaplan–Meier method was used for univariate survival analysis, and the log-rank test was used to assess the difference between survival curves. Spearman’s correlation coefficient was used to determine the correlation between the expression levels of KSR2 and 14–3-3ζ. Univariate and multivariate tests were carried out using two-sided Cox univariate analyses. Data are presented as the mean ± standard deviation (SD) or the mean ± standard error of the mean (SEM). Differences were considered statistically significant at p values < 0.05.