Osteosarcoma (Saos-2, U2OS, and SJSA), rhabdomyosarcoma (RH30, RH3 and RD2), leiomyosarcoma (SK-LMS-1), human foreskin fibroblast (HFF), and human skeletal muscle myoblast (HSMM) cell lines were purchased from American Type Culture Collection (ATCC). CW9019, a rhabdomyosarcoma cell line, was a gift from Dr. Fred Barr (Department of Pathology, University of Pennsylvania). All cell lines were maintained in 1× DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicilin/streptomycin/amphotericin B (Fisher Scientific International) at 37°C, aired with 5% CO2. HSMM cells were grown in SkBM-2 basal medium supplemented with SkGM-2 singleQuots kit according to the manufacture's protocol (Cambrex Bio Science, Walkersville, MD, USA).
Cancer tissue microarray immunohistochemistry
To examine whether Stat3 is activated in rhabdomyosarcoma, osteosarcomas, and other soft-tissue sarcomas, we stained osteosarcoma (n = 113), rhabdomyosarcoma (n = 64) and other soft-tissue sarcoma (n = 87) tissue samples on tissue microarray slides from different providers (Biopathology Center of Columbus Children Research Institute, Biomax, and Cybrdi) using immunohistochemistry with a p-Stat3(Y705)-specific monoclonal antibody (Cell Singling Tech., Danvers, MA). The immunohistochemistry method was described previously [5, 15]. Nuclear p-Stat3 expression levels were scored as 0, 1, 2, and 3 according to the immunohistochemical staining intensity. The nuclear staining intensity was scored on the following scale: 0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining. Since all the normal tissues stained were scored as 0 and occasionally 1, samples stained with scores 0 or 1 were considered as negative, whereas sarcoma samples with scores 2 and 3 were graded as positive. The intensity of immunostaining was evaluated only when more than 50% nuclei showed p-Stat3 expression. Scoring of immunostaining intensity was completed by two to three independent observers (F. H., G. C., and J. L.). Discrepant scores between the two or three observers were rescored to arrive at a single final score. Light microscopic images were documented using a LEICA DM-4000B fluorescent microscope (Leica Microsystems, Bannockburn, IL) with an attached Diagnostic RT-KE 2 MP digital camera (Diagnostic Instruments, Sterling Heights, MI).
Cells were collected at 4°C in cold harvest buffer supplemented with proteinase inhibitor cocktails and spun down at 3000 × g for 5 min. Cell pellets were lysed in RIPA lysis buffer as described previously . Protein concentrations were quantitated using BCA protein assay kit from Pierce, Inc. (Rockford, IL) according to the manufacture's protocol. Fifty or 100 μg of cellular proteins were resolved on 10% PAGE gels in electrophoresis buffer and transferred to Hybond™-p membrane (Amersham Biosciences, Piscataway, NJ) using transfer buffer with a constant 100 V. The membranes were then blocked using 5% nonfat dry milk in TBST (Tris-HCl, pH7.5, Tween, 0.1%) for 30 min at room temperature (RT) and were incubated with primary antibody over night at 4°C or for 1 hour at RT using concentrations recommended by the manufacture. The membranes were washed three times in 1× TBST for 5 min each time. Proteins of interest were visualized using an ECF™ western blotting kit (Amersham Biosciences, Piscataway, NJ) according to the manufacture's protocol. Incubation of secondary antibody and anti-fluorescein were carried out both in presence of 1× TBST with 2% nonfat dry milk. The fluorescent signals were scanned and documented using a Storm 860 scanner (Molecular Dynamics, Sunnyvale, CA). Antibodies were purchased separately and used for Western blots of FLAG (Sigma, St. Louis, MO, USA), GAPDH (Chemicon International, Temecula, CA), Stat3, and p-Stat3 (Y705) (Cell Signaling Tech., Danvers, MA).
Transduction of dominant negative Stat3 Y705F in cancer cells
The construction and infection of recombinant Adenovirus/CMV-dnStat3 Y705F (rAd/dnStat3) is described previously [14, 16]. DnStat3 was generated from Stat3 by changing the tyrosine at position 705 into phenylalanine. Its protein product cannot be activated through tyrosine phosphorylation that is crucial for dimerization. The clone is tagged with a FLAG marker. About 2 × 105 U2OS, SaoS2, SJSA, RD2 and RH30 cells were transduced with rAd/dnStat3 or a negative control viral vector, rAd/CMV-eGFP (rAd/eGFP) (Applied Viromics, Fremont, CA) with multiplicities of infection (moi) of 400, 100, and 10 based on TCID50 using 293T cells. For cell growth experiments, cells in 5 random fields of view (100× magnification) were enumerated on days 2, 4, and 6 post-infection of rAd/eGFP and rAd/dnStat3. Cell growth rates were presented as percentages of untransduced controls. Each data point was averaged from triplicate experiments.
Treatment of STA-21 and cell viability assay
Approximately 5000 RD2 and RH30 cells were grown in 100 μl 10% FBS-supplemented DMEM medium in 96-well flat-bottomed plates overnight. Treated cells were exposed to STA-21 (30 μM) that was dissolved in dimethyl sulfoxide (DMSO) before being added to the medium. Cell viability was analyzed by the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] (Sigma, St. Louis, MO, USA) assay in three replicates. At the endpoint, cells were treated with MTT (1 mg/ml) for 3–4 hours. Colorimetric quantification was determined by an EL808 Ultra Micro-plate Reader (Bio-Tek Instruments, Inc) after the addition of formazan dissolved in 25% N, N-dimethylformamide and 10% SDS under light-proof conditions overnight.
Caspases 3, 8, and 9 immuno-fluorescent staining and acridine orange staining
1 × 105 cells (U2OS or RD2) were seeded on sterile coverslips in a 6-well plate overnight. The cells were transduced by either rAd/eGFP or rAd/dnStat3 for 3 or 4 days and then fixed using methanol/acetone (v:v = 1:1). Three washes followed the fixation using 1× PBS buffer. During the third wash, the coverslips were transferred to a new 6-well plate. For immuno-fluorescent staining, the cells were blocked in 1× PBS with 10% normal horse serum for 1 hour and incubated with primary rabbit antibodies that recognize cleaved-caspase-3 (Asp175), cleaved-caspase-8 (Asp374), or cleaved-caspase-9 (Asp330) (Cell Singling Tech., Danvers, MA) with 1:100, 1:50, and 1:100 dilutions, respectively. Excess antibodies were removed using 3 washes of 1× PBS with constant agitation, 10 minutes for each wash. Secondary goat anti-rabbit IgG(H+L) Alexa FluoR 594 antibody (Invitrogen, Carlsbad, CA) (1:1000 dilution) was incubated with 1% bovine serum albumin (BSA) in 1× PBS for 1 hour at RT. Unbound antibody was washed off three times using 1× PBS. Nuclei were counter-stained using 4'-6-Diamidino-2-phenylindole (DAPI) (100 ng/ml) in distilled H2O for 5 min and then rinsed three times with 1× PBS, 10 min for each wash. The cleaved caspase positive cells were scored from three independent fields of view (100× magnification) and presented in averaged percentages of total cells (DAPI staining) with standard deviations from triplicate experiments.
Acridine orange staining was previously described. The cells were incubated with 1 mg/ml acridine orange (Sigma, St. Louis, MO) for 15 min before 3 washes of 1× PBS. The fluorescence and phase-contrast microscopic photographs were documented using LEICA DM-IRB inverted fluorescent microscope (Leica Microsystems, Bannockburn, IL) with an attached Diagnostic RT-SE6 monochrome digital camera (Diagnostic Instruments, Inc, Sterling Heights, MI).