Flow cytometric determination of apoptosis by annexin V/propidium iodide double staining.

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1 Supplementary materials and methods Flow cytometric determination of apoptosis by annexin V/propidium iodide double staining. Cells were analyzed for phosphatidylserine exposure by an annexin-v FITC/propidium iodide double-staining method described by Vermes et al (1). The cells 8 days after transfection of sirnas were collected and subjected to the analysis. A minimum of 5,000 cells were then analyzed by FACScan with Cell Quest software (Beckton Dickinson) for acquisition and analysis. DNA Fragmentation Assay DNA fragmentation assay was performed as previously described (2). The WE-68 cells transfected with 100 nm of siscr or sibpefi, including floating cells, were scraped and collected everyday after the transfection. The cells were dissolved in 100 µl of cell-lysis buffer [10 mm Tris (ph 7.5) and 10 mm EDTA (ph 8.0) and 0.5% Triton X-100] and then incubated on ice for 10 min. The lysates were then centrifuged at 16,000 g for 5 min at 4 C. The supernatant was recovered and treated with RNase A (Roche, Indianapolis, IN, USA) for 1 hr at 37 C and then with Protease K (Roche) for 1 hr at 50 C. DNA was precipitated by adding isopropanol and 5 M NaCl to the mixture, and the precipitates were resuspended in TE [10 mm Tris (ph 8.0)/1 mm EDTA]. The samples were electrophoresed on 1.2% agarose gels, which were stained with 0.5 mg/ml ethidium bromide (Sigma, St Louis, MO, USA) for 10 min to visualize the DNA ladder. For immunofluorescence microscopy, the cell cultures on the chamber slides were fixed with 4% formaldehyde in PBS for 10 min at room temperature and permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS as described previously (matsumoto). Filamentous actin was stained for 1 hr with 10 µl of rhodamine-phalloidin (Molecular Probes) in 200 µl PBS (0.3 µm final concentration), and washed extensively with PBS. The nuclei were stained with DAPI (4,6-diamidino- 2-phenylindole dihydrochloride; Molecular Probes) at 0.1 µg/ml in PBS. The cells were examined

2 using fluorescence microscope. Immunohistochemistry and statistical analysis Immunohistochemical studies of Skp2 and p27 in 25 primary EFTs cases were performed using the standard streptavidin-biotin-peroxidase method (Histofine SAB-PO kit, Nichirei, Tokyo, Japan) on formalin-fixed, paraffin-embedded tissues as described previously (2). The primary antibodies against Skp2 (Santa-Cruz) and p27 (Kip1; Transduction Laboratories) were used at dilutions of 1:100 and 1:500, respectively. The p27 labeling index (p27 LI) and the Skp2 labeling index (Skp2 LI) were defined as the percentage of tumor cells displaying immunoreactivity for p27 and Skp2, respectively, as described previously (2). Both p27 and Skp2 expressions were graded as negative when less than or equal to 10% of the tumor cells were stained, and they were graded as positive when greater than 10% of cells were stained. Associations between the variables were tested by Fisher s exact test. All statistical differences were deemed significant at the level of P < Patient population A series of 25 patients preoperatively diagnosed as EFTs at Kyushu University Hospital, the National Kyushu Cancer Center and the National Kyushu Medical Center, between 1982 and 2004 was used in this study. The study was in agreement with the World Medical Associations Declaration of Helsinki in 1975, revised in A signed letter of informed consent was obtained from each patient. The diagnosis of EFTs was made based on the histological features. There were 13 male and 12 female patients, ranging from 8 to 75 years old, with a median age of 21 years. All cases presented with primary EFTs and were treated with systemic chemotherapy based on the VACA or VAIA protocol as described previously (2). The average follow-up period was 40.5 months from the time of diagnosis (range, 5 to 119 months). 2

3 Supplementary references for materials and methods 1. Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods, 184: 39-51, Matsunobu, T., Tanaka, K., Matsumoto, Y., Nakatani, F., Sakimura, R., Hanada, M., Li, X., Oda, Y., Naruse, I., Hoshino, H., Tsuneyoshi, M., Miura, H., and Iwamoto, Y. The prognostic and therapeutic relevance of p27kip1 in Ewing's family tumors. Clin Cancer Res, 10: ,

4 Supplementary Figure Legends Supplementary Figure 1. (A) DNA fragmentation assay. DNA was extracted from WE-68 cells treated with 100 nm of sirnas for the required time as indicated in the figure and electrophoresed in 1.2% agarose gel as described in Methods. Lane M, the 100 bp DNA size marker; Lane P, the positive control sample for DNA ladder from WE-68 cells treated with Doxorubicin (100 ng/ml) for 24 hrs. (B) Western blot analysis for EWS-Fli1, PARP and actin expression. Lysates from WE-68 cells treated with 100 nm of sirnas for the required time as indicated in the figure were prepared and subjected to western blot analysis. Filled arrowhead indicates the uncleaved form, while open arrowhead indicates the cleaved form of the PARP protein. P; the positive control sample for the cleaved PARP from WE- 68 cells treated with Doxorubicin (100 ng/ml) for 24 hrs. (C) Western blot analysis for EWS-Fli1, p27 and actin expression. Lysates from WE-68 cells treated with 100 nm of sirnas for the required time as indicated in the figure were prepared and subjected to western blot analysis. (D) Relative amount of EWS-Fli1 mrna to GAPDH mrna. RNAs from EFT cells treated with 100 nm of sirnas for the required time as indicated in the figure were prepared and subjected to real-time quantitative PCR as described in Materials and methods. (E) Annexin V-propidium iodide staining to examine apoptosis and necrosis. Apoptotic cells and necrotic cells in WE-68 cells treated with siscr or sibpefi for 8 days were determined by annexin V/propidium iodide labeling as described in Materials and Methods. Supplementary Figure 2. (A) Microscopic photographs of RD-ES and SK-N-MC 72 hrs after the transfection of sirnas. The filamentous actin was stained with rhodamine-phalloidin and the nuclei were stained with DAPI. The cells stained were examined using a fluorescence microscope. (B) The percentages of WE-68 cells which were positively stained for SA β-gal are shown. The cells transfected with 100nM of sirnas for 8 days were analyzed for SA β-gal staining. White and black bars represent the percentages of positively stained cells treated with siscr and sibpefi, respectively. 4

5 Bars represent S.D. (C) The number of PNKT-1cells was counted over the entire 8 days after the transfection of sirnas. Symbols represent mean value of the cell number of siscr-treated cells (circle) and sibpefi-treated cells (square). Bars represent S.D. (D) Microscopic photographs of PNKT-1 after the transfection of sirnas. Bars indicate 100µm. Supplementary Figure 3. Immunohistochemical staining of two consecutive sections of p27 and Skp2 in EFTs. Left and right columns represent microscopic photographs of an Skp2-weak case and an Skp2-strong case, respectively. A and B, H-E staining. C and D, p27 immunostaining. E and F, Skp2 immunostaining. Bars indicate 50µm. The left column shows microscopic photographs of a representative tumor which arose in 35-year-old man was p27-positive but Skp2-negative (A, C and E). The patient has remained well for 119 months since being diagnosed. In the case, the p27 LI was 37.3% and the Skp2 LI was 5.7%. Another tumor from a 12-year-old girl was p27-negative but Skp2- positive (B, D and F). The patient died of multiple tumor metastasis 30 months after her first admission. In this case, the p27 LI was 3.04% and the Skp2 LI was 13.4%. 5