Cell lines and culture conditions
The HCC cell lines HLF, SK-Hep1, and Bel7402 were obtained from the Hepatic Surgery Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China. HEK293 cells were purchased from the China Center for Type Culture Collection (Wuhan, China). The cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 incubator at 37 °C.
To establish hmox1 or 14–3-3ζ overexpressing cell lines, the human cDNA was cloned into the pBABE-puro retroviral vector (plasmid 1765, Addgene, Cambridge, MA, USA). To establish stable 14–3-3ζ, hmox1, and STAT3 knockdown or control cells, the target short hairpin RNA (shRNA) sequences and one non-targeting sequence (negative control, NC) were selected and cloned into the pLKO.1 vector (pLKO.1 puro, plasmid #8453, Addgene). Viral packaging and transduction were performed as previously described . The target shRNA sequences are listed in Additional file 1: Table S1. Mammalian expression plasmids (pcDNA3.1) for Flag- or HA-tagged HO-1 and its mutants, HA-tagged 14–3-3ζ were created according to a standard PCR-based targeted mutagenesis protocol and were verified by sequencing (Sangon Biotech Co., Ltd., Shanghai, China). The sequences of the primer pairs used to generate each mutant are listed in Additional file 1: Table S2. STAT3 luciferase reporter plasmids were purchased from Qiagen. Mammalian expression plasmids for JAK1, and its mutants, JAK2 and its mutants, STAT3 and its mutants were constructed by standard molecular biology techniques and purchased from GeneChem (Shanghai, China).
Mass spectrometry (MS)
Whole cell lysates from 293FT cells transient transfected with FLAG-HO-1 or FLAG-vector were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5% NP-40, 1 mM EDTA and protease inhibitor cocktails) and applied to an IP lysis buffer equilibrated FLAG affinity gel (Sigma-Aldrich, St. Louis, MO, USA) in 4 °C for 4 h. After binding, the affinity gels were washed with the lysis buffer for five times and the pellets were resuspended in SDS sample buffer and boiled for 8 min. The final eluted proteins were separated by SDS-PAGE followed by silver staining of the gel. There was a great difference in the bands between the FLAG-HO-1 group and the control FLAG-vector group among the molecular weight 15kd-40kd region of the gel. The relevant bands (from 15kd~40kd) were excised from the gel and cut into pieces. For in-gel tryptic digestion, gel pieces were destained in 50 mM NH4HCO3 in 50% acetonitrile (v/v) until clear. Gel pieces were dehydrated with 100 μl of 100% acetonitrile for 5 min, the liquid removed, and the gel pieces rehydrated in 10 mM dithiothreitol and incubated at 56 °C for 60 min. Gel pieces were again dehydrated in 100% acetonitrile, liquid was removed and gel pieces were rehydrated with 55 mM iodoacetamide. Samples were incubated at room temperature, in the dark for 45 min. Gel pieces were washed with 50 mM NH4HCO3 and dehydrated with 100% acetonitrile. Gel pieces were rehydrated with 10 ng/μl trypsin resuspended in 50 mM NH4HCO3 on ice for 1 h. Excess liquid was removed and gel pieces were digested with trypsin at 37 °C overnight. Peptides were extracted with 50% acetonitrile/5% formic acid, followed by 100% acetonitrile. Peptides were dried to completion and resuspended in 2% acetonitrile/0.1% formic acid. Then, the resulting tryptic peptides were subjected to liquid chromatography-tandem MS (LC-MS/MS) sequencing. The masses of peptides were identified by time-of-flight analysis using a model 4700 protein analyzer (Applied Biosystems, Franklin Lakes, NJ, USA). Data obtained by MS and MS/MS were queried against the Swiss-Prot database (Homo sapiens).
For immunofluorescent microscopy after the indicated treatments, cells were seeded onto glass slides for 24 h, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 for 15 min. The slides were then incubated with a primary antibody in blocking solution overnight at 4 °C in a humidified chamber. The slides were then washed three times with phosphate buffered saline (PBS) and incubated with one or both of two secondary antibodies (from different species) for 1 h at room temperature in a humidified chamber. Finally, the coverslips were incubated with 40, 60-diamidino-2-phenylindole (Sigma-Aldrich) for 15 min, and images were obtained by phase-contrast and confocal microscopy.
Immunoblotting, co-immunoprecipitation (co-IP) and protein ubiquitination assays
Immunoblot, co-IP, and in vivo ubiquitination assay were carried out as described previously .
Cell proliferation assay and colony formation assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China). Cells were plated in 96-well plates at the indicated density and cultured in complete medium. At the indicated times, 10 μL of CCK-8 was added to 90 μL of culture medium per well for 2 h at 37 °C, and the plate was read using an Elx 800 enzyme-linked immunosorbent assay plate reader (Bio-Tek, Winooski, VT, USA) at 450 nm. Each cell line was assayed in triplicate wells, and the experiment was repeated three times. For the colony formation assay, 1000 to 3000 cells were plated into each well of a 6-well plate and incubated at 37 °C for 2 weeks. The colonies were fixed and stained with a dye solution containing 0.1% crystal violet and 20% methanol, and the number of colonies was counted. All assays were replicated three times.
Transfection and luciferase reporter assays
Small interfering RNA (siRNA) and si-control were purchased from Ribobio (Guangzhou, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for the transient transfection of small RNA oligos and plasmids according to the manufacturer’s instructions. siRNA target sequences are listed in Additional file 1: Table S3. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega) according to manufacturer’s protocols after transfection. All reporter assays were repeated at least three times.
Quantitative real-time PCR assay
Total RNA was extracted using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) and reverse transcription was performed using the PrimeScript® RT reagent Kit (Takara Bio, Dalian, China) according to the manufacturer’s protocol. Quantitative real-time PCR was carried out using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the SYBR Green Supermix kit (Takara Bio) according to the manufacturers’ instructions. All of the gene expression levels were normalized to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each reaction was repeated independently at least three times in triplicate. The primers used are summarized in Additional file 1: Table S4.
Male Balb/c athymic nude mice (3–4-week-old) were housed under specific pathogen-free conditions and cared for according to the institutional guidelines on animal care. The mice were randomly divided into the indicated groups (5–8 mice/group) before inoculation. For subcutaneous injection, 2 × 106 tumor cells were suspended in 100 μL of serum-free DMEM, and then injected subcutaneously into the flank of the nude mice. Tumor development in the mice was observed every 3 days. The mice were sacrificed at a defined endpoint. Tumors were removed, photographed, measured, and weighed. The average volume and weight of the tumors were calculated.
Cell viability assay, AnnexinV/Propidium iodide staining, and TUNEL assay
Cell viability was analyzed by Trypan Blue Staining Cell Viability Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) and counted by Nexcelom Cellometer Auto X4 Cell Counter (Nexcelom Bioscience, Massachusetts, USA). All viability experiments were repeated as three independent experiments, and mean percentage of cell survival was calculated along with SD. Student’s t test was used for calculating statistical significance. Cell apoptosis was done by Annexin V-fluorescein isothiocyanate (FITC)/propidiumiodide (PI) double staining using flow cytometry analysis. Cells were incubated with Annexin V-FITC and PI for 15 min at room temperature in the dark. The samples were processed to the FASCcan Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). Apoptosis in tumor was examined by TUNEL assay using TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Tissue sections were dewaxed and rehydrated according to standard protocols. TUNEL staining was performed according to the protocols from the manufacturers. Apoptotic cells were photographed with microscope.
Reagents and antibodies
Cycloheximide and MG132 were purchased from Calbiochem (San Diego, CA, USA). Antibodies were obtained from the following sources: monoclonal anti-HO-1 (ab52947, Abcam, Cambridge, UK; used for Immunoblot, co-IP), polyclonal anti-HO-1 (10701–1-AP, Proteintech, Chicago, IL, USA; used for Immunofluorescent staining), anti-β-actin (A5316, Sigma-Aldrich), monoclonal anti-Flag (F1804, Sigma-Aldrich), monoclonal anti-HA (H9658, Sigma-Aldrich), polyclonal anti-Flag (F7425, Sigma-Aldrich), anti-14-3-3β (sc-25,276, Santa Cruz Biotechnology, Dallas, TX, USA), anti-14-3-3θ (sc-59,414 Santa Cruz Biotechnology,), anti-14-3-3η (sc-423,749, Santa Cruz Biotechnology), anti-14-3-3γ (12381-1AP, Proteintech), anti-14-3-3ε (11648–2-AP, Proteintech), anti-14-3-3ζ (15222–1-AP, Proteintech), anti-ERp72(66365–1-Ig, Proteintech), anti-STAT3 (#12640, Cell Signaling Technology, Beverly, MA, USA), anti-JAK2 (#3230, Cell Signaling Technology), anti-pY705-STAT3 (#9145, Cell Signaling Technology), anti-pS727-STAT3 (#9134, Cell Signaling Technology), and and anti-ubiquitin (#3936, Cell Signaling Technology). Fluorochrome-conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). Agarose-immobilized anti-FLAG (M2, Sigma-Aldrich) was used for IP. The antibodies used as negative controls for IP included normal mouse IgG (sc-2025, Santa Cruz Biotechnology) and normal rabbit IgG (sc-2027, Santa Cruz Biotechnology).
Data were statistically analyzed using Graphpad Prism 6 software (GraphPad Software, San Diego, CA, USA), and presented as mean ± s.d., Unpaired two-tailed t-test or ANOVA test was utilized to analyze the difference between the two groups. Three independent experiments were performed to guarantee reproducibility of findings. A P-value < 0.05 was considered statistically significant.