Materials and Methods
Cell Culture, Reagents and Plasmid Construction
MCF7, MDA-MB-231, and HEK293T cells were obtained and characterized by a cytogenetic analysis by American Type Culture Collection (ATCC) and maintained in this lab according to the recommendation of ATCC. The cell lines were characterized in this lab by morphological analysis before using for experiments. The v-Src/NIH3T3 cell line was a gift from Dr. H Yu at City of Hope Comprehensive Cancer Center, California, USA and was characterized by morphological analysis in this lab according to her recommendation. The stable cell line for depletion of PTPMeg2 by shRNA was generated in this lab based on MCF7 and characterized by morphological analysis and the expression of targeted gene was characterized by a Western blot. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in 5% CO2 astrosphere in 37°C. The mouse hepatic cell lines STAT3−/− (KO) and STAT3+/+ (WT) derived from STAT3 conditional knockout and wild-type mice were also cultured in DMEM medium.
Anti-sera against PTPMeg2 were generated by immunizing rabbits with purified GST-PTPMeg2 (1-1779) proteins in ZJ Zhao's lab. Anti-Myc (9E10), anti-HA (F-7), anti-GFP (FL), anti-pSTAT3(Tyr705, B-7), anti-pSTAT3(Ser727), anti-STAT3 (F-2) and anti-STAT3 (C-20) antibodies, and protein G/A plus agarose beads were purchased from Santa Cruz Biotechnology, and anti-β-Actin (AC-15) antibody from Sigma. MG132 and leupepstin were purchased from Amresco (AMRESCO Inc. OH), and human recombinant IL-6 and IL-6 receptor from B&D (B&D SYSTEM, USA). Plasmids including GST-STAT3, pXJ40-STAT3, and its deletions were kindly provided by Dr. Xinmin Cao. Expression vectors for human PTPMeg2 and PTPMeg2C515S or deletion mutants were constructed based on pcDNA3.1-Myc or pEFneo-Myc. Other plasmids involved in this study were stored in the lab.
MCF7 cells were plated in 24-well plates the day before transfection. An amount of 0.1 μg of reporter plasmid pAPRE-luc or M67-luc together with 5 ng of an internal control plasmid pRL-TK was transfected. Constructs expressing STAT3, PTPMeg2 and its mutants were co-transfected at an amount of 0.4 μg per well. To deplete endogenous PTPMeg2, 0.8 μg of vectors with shRNA targeting PTPMeg2 (shRNA1: 5'-GATCCGGAAAGGCATTGTAAATTCAAGAGATTTACAATGCCTTCCTTCCTTA-3', shRNA2: 5'-GATCCGCAAGGAATCTATGAGGAATTCAAGAGATT CCTCATAGATTCCTTGCTTA-3', shRNA3: 5'-GATCCCTAGAGTGAAGCT AACAATTCAAGAGATTGTTAGCTTCACTCTAGTTTA-3') in pSilencer-4.1 was transfected. Twenty-four hours after transfection, luciferase assays were performed with a dual-luciferase reporter assay system (Vigofect Inc. Beijing, China) and the luciferase activity was normalized by firefly against the renilla luciferase activity.
Western Blot, GST Pull Down and Immunoprecipitation Assay
Proteins were analyzed by SDS-PAGE and Western blot. For immunoprecipitation experiments, HEK293T cells grown in 60 mm dishes were transfected with indicated expression plasmids and were lysed in cell lysis buffer (20 mM Tris-Cl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 10 mM MgCl2, 0.5% NP40, 10% glycerol, 1 mM DTT, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) for 30 min on ice, and then the lysates were centrifuged at a maximum speed for 15 min. Supernatants of cell lysates were incubated with 2 μg of indicated antibodies overnight at 4°C, and 30 μl of protein IgG/A agarose plus beads were added for binding for 4 h at 4°C. Beads were washed with cell lysis buffer (containing 0.1 mM vanadium) 4 times and bound proteins were eluted with 2 × loading sample buffer and analyzed by Western blot with indicated antibodies. For GST-pull down assays, the procedure was similar to that in immunoprecipitation experiments except that GST beads were used and washed by PBST buffer.
In Vitro Dephosphorylation Assay
GST-PTPMeg2 WT and GST-PTPMeg2CS proteins were expressed and purified as described previously. Phosphorylated Flag-STAT3 was prepared from HEK 293T cells transfected with Flag-STAT3 for 48 h and then stimulated with IL-6 for 30 min. Purified phosphorylated Flag-STAT3 was incubated with GST and GST-PTPMeg2 WT or GST-PTPMeg2CS proteins at 37°C for 30 min with PTPase reaction buffer (25 mM Tris-Cl, pH 7.5, 2.5 mM EDTA, 1 mM EGTA, 1 mg/ml BSA, 10 μM TPCK, 1 mM Benzamidine, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). The dephosphorylation reaction was terminated by directly boiling. Proteins were separated with SDS-PAGE and analyzed with an anti-pSTAT3 and anti-Flag antibodies by a Western blot.
Cells were seeded on glass coverslips for 24 h and subjected to serum-starvation for 18 h before treatments with IL-6 for 30 min. Cell was fixed in 4% paraformaldehyde for 20 min at 4°C and permeabilized in 0.3% Triton X-100 for 15 min. Cells were blocked with 10% normal rabbit serum at room temperature and incubated in primary antibody overnight at 4°C. Primary antibodies used were an anti-PTPMeg2 or an anti-Flag antibody. Secondary antibodies used were FITC-conjugated and TRITC-conjugated IgG. Nucleus was stained with DAPI.
Cell Growth Experiment
MCF7 cells stably transfected with the shPTPMeg2 plasmid, or MDA-MB-231 or mouse hepatic STAT3 KO cells infected with an adenovirus for over-expression of PTPMeg2, were seeded on 96-well plates at a density of 1000 cells/well in triplicate. Cells were cultured for different times and 5 g/L of MTT was added 4 h before termination of cell growth. The purple blue sediment was dissolved in 150 μl of DMSO before harvest. The relative optical density (OD)/well was determined at wavelength of 570 nm in a WELLSCAN MK3 ELIASA (Lab systems, Dragon, Finland) using a 630 nm reference filter. Cell growth curve was drawn according to the average of OD570-OD630.
Xenograft Tumor Model
Exponentially growing MCF7 cells were stably transfected with the PTPMeg2 shRNA vector and MDA-MB-231 were infected with the adenovirus or retrovirus and v-Src/NIH3T3 cells with the adenovirus for over-expression of PTPMeg2. Cells were suspended in 1 ml physiological saline for preparation of injection into mice. BALB/c-nu/nu mice at 5 weeks of age were subjected to bilateral subcutaneous injections with 1.0 × 107 (MCF-7) or 5.0 × 106 (MDA-MB-231) or 5.0 × 105 (v-Src/NIH3T3) cells in a volume of 0.1 ml saline. Tumor volume defined as (length × width2)/2 was measured every two days with a caliper up to study termination. Tumor growth curves were drawn according to the average of tumor volumes (mm3). All animal experiments were performed in accordance with the institutional animal experiment guidelines.
Patient Samples and Immunohistochemistry
The formalin-embedded tissue samples from 73 patients with breast carcinomas diagnosed between 2008 and 2010 were obtained from the Surgical Pathology in the TangShan People's Hospital. All breast cancer specimens from female patients were obtained from clinical surgery. Information of age, histological type, differentiation grade, and lymph node metastasis of breast carcinomas were obtained from the Surgical Pathology Files in the hospital. The clinicopathological diagnosis on the tumor status was evaluated by the clinical pathologists in the hospital. All samples used in the study were approved by the TangShan People's Hospital Ethical Committee under the guidance of tissue collection procedure with informed consent.
Sections of formalin-fixed breast carcinoma tissues were treated with 0.3% hydrogen peroxidase/methanol and incubated with primary antibodies followed by incubation with secondary antibodies (biotin-labeled; Santa Cruz) and third antibodies (peroxidase-labeled; Santa Cruz). Samples were developed using DAB as substrates (Santa Cruz). Scoring criteria for tumor degrees as reported previously were used. Briefly, the grade was classified as 0 for negative, 1 for weak (< 10%), 2 for moderate (10–25%), 3 for strong (26–75%) and 4 for very strong (> 75%) staining according to percentage of positively staining cells. The staining index was subsequently obtained by multiplication of the proportion and intensity and calculated index was finally assessed by a simplified score (score 0, index 0–1; score 1, index 2–4; score 2, index 6–8; score 3, index 9–12). Samples with staining score of at least 1 were classified as positive staining, score 2 and 3 were strong positive staining. The percentage of pSTAT3 nuclear positive cells were used to classify the grade of its expression as negative (< 5%), weak (6–50%), and strong (51–100%).
All experiments were repeated at least three times. The Student's t test was used to evaluate the significance of differences between experimental and control groups. Data were analyzed by one-way ANOVA with SPSS13.0 (SPSS, Int, Chicago, IL). Frequencies of PTPMeg2, STAT3 and pSTAT3 expressions among cancer samples were analyzed by the x2 test with a modification by the Fisher's exact test to account for frequency values < 5. The correlation between protein levels was evaluated by the pair-wise Pearson correlation coefficient and by bi-dimensional hierarchical clustering. All Ps reported were two-sided. Significance was defined at the level of P ≤ 0.05.
Breast Cancer Res. 2012;14(2):R38 © 2012 BioMed Central, Ltd.
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