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Sustained Loss of a Neoplastic Phenotype by Brief Inactivation of MYC
Meenakshi Jain, Constadina Arvanitis, Kenneth Chu, William Dewey, Edith Leonhardt, Maxine Trinh, Christopher D. Sundberg, J. Michael Bishop, and Dean W. Felsher

Supplementary Material

Materials and Methods

Generation of cell lines: Cell suspensions were generated by gently teasing tumors between frosted slides. Cell lines were then established by the culture of these tumor cells on treated plates maintained in DMEM containing 10% FBS and 1% Penicillin-Streptomycin at 37°C in humidified atmosphere with 5% CO2.

MYC Inactivation in vivo: Mice were initially injected intraperitoneally with 100 Greek Letter Mug of dox. Dox was also added to their drinking water, at a concentration of 100 Greek Letter Mug/ml changed once per week.

CVTL: Experiments were performed on cells plated at a density of 2 x 103. The cells were incubated for 18 hours to allow for attachment. The flask was then capped tightly and placed onto the microscope stage inside an incubation chamber maintained at 37°C. Fifty different fields in the flask containing one to three cells were examined. Phase-contrast images at 200X were collected with the CVTL system for 29.8 hours. Then, dox (20 ng/ml) was added to the medium, and images were acquired of the same 50 fields every 10 minutes over a period of 89.7 hours until the dox treatment was terminated. The dox was removed by aspirating off the medium, rinsing the flask three times with PBS, and incubating with 5 ml fresh medium for 15 min. The medium was replaced with another 5 ml of fresh medium and the pH was adjusted by gassing with 5% CO2 for ~1 minute. The flask was then capped tightly and returned to the CVTL microscope stage. Images were collected for another 95.7 hours from the same 50 fields (S1).

Histology: Tissues were fixed in 10% buffered formalin and 5 �M paraffin sections were prepared and stained with Hematoxylin and Eosin.

Immunocytochemistry: Tumor-derived cell lines were seeded in multi-chambered tissue culture slides (Becton Dickinson Lab, NJ). In some cases, MYC transgene expression was inactivated by treating the cells with dox at a final concentration of 20 ng/ml for 2 days. MYC expression was detected using primary antibody c-MYC A-14 at a dilution of 1:50 (Santa Cruz, CA).To detect Osteopontin we used antibody LF124 at a dilution of 1:100 Immunocytochemistry was performed using the Vectastain Elite ABC system and DAB substrate (Vector Lab, CA). Alkaline Phosphatase (ALP) activity was measured with histochemical analysis kit (Sigma, MO) exactly as described by the manufacturer. Positive staining was blue staining of >50% of cells.

TUNEL: Apoptic cells were detected by the TUNEL assay in situ death detection kit (Roche Diagnostic, IN) as described by supplier. Cells were counterstained with DAPI (Vector Lab, CA).

References: S1. H. B. Forrester, C. A. Vidair, N. Albright, C. C. Ling, W. C. Dewey, Cancer Res. 59, 931 (1999).

Supplemental Figure 1.MYC- induced invasive osteogenic sarcomas. (A) Gross pathology of a transgenic mouse with an osteogenic sarcoma of the calvarium (see arrow), and metastatic lesions in the lung and liver. (B) Microscopic pathology of tumors reveals immature osteoblasts with the formation of disorganized osteoid (see arrows). Tumors were highly metastatic to the (C) liver, (D) retroperitoneum, and the (E) lung. Representative pathology from one of over 10 tumors examined is shown. Size bars equal 50 �M.

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Supplemental Figure 2. Inactivation of MYC causes regression and differentiation of osteogenic sarcomas. (A) Prior to MYC inactivation mice bearing transplanted tumor cells exhibited ascites, enlargement of spleen and liver, and tumors adherent to the gastrointestine (see arrows). (B) After MYC inactivation, tumors regressed. Multiple calcified masses were found attached to the gastrointestine (arrows). Size bars equal 50 �M.

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Supplemental Figure 3.MYC inactivation followed by reactivation in vitro causes apoptosis of tumor cells. (A) Prior to MYC inactivation, tumor cells were proliferative. (B) After MYC inactivation, the rate of cell division was reduced, and cell size increased. (C) The consequences of MYC reactivation are shown in one representative cell. After 12 hours of MYC reactivation there was no apparent change in cell morphology. (D) After 12.7 hours, the cell became rounded. (E) After 12.8 hours there was evidence for blebbing. (F) After 14.8 hours the cell had died. (G) After 17.7 hours the membrane had ruptured. Size bars equal 10 microns.

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Supplemental Figure 4.MYC inactivation causes differentiation and reactivation causes apoptosis and the loss of tumor cells in primary transgenic tumors. Serial biopsy specimens were taken from a transgenic mouse with a primary osteogenic sarcoma in its tail prior to treatment with dox, after treatment with dox for 10 days or after removal of dox for 6 days. (A�C) Histology, (D�F) TUNEL staining and (G�I) DAPI staining. Similar results were seen for an additional primary osteogenic sarcoma. Size bars equal 50 �M.

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