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1 Supplementary methods: Primers sequences used in real-time PCR analyses: β-actin F: GACCTCTATGCCAACACAGT β-actin [11] R: AGTACTTGCGCTCAGGAGGA MMP13 F: TTCTGGTCTTCTGGCACACGCTTT MMP13 R: CCAAGCTCATGGGCAGCAACAATA MMP7 F: TGAATTTGGCCACTCTCTGGGTCT MMP7 R: TCTGAATGCCTGCAATGTCGTCCT MMP12 F: TAGAAGCAACTGGGCAACTGGACA MMP12 R: ACCGCTTCATCCATCTTGACCTCT MMP3 F: ACCAACCTATTCCTGGTTGCTGCT MMP3 R: ATGGAAACGGGACAAGTCTGTGGA MMP10 F: GACCCCAGACAAATGTGATCCT MMP10 R: TTCAGGCTCGGGATTCCA MMP8 F: AATCCTTGCCCATGCCTTTCAACC MMP8 R: CCAAATTCATGAGCAGCCACGAGA MMP20 F: AGCAAGAGAGGAGATGAAGGTGCT MMP20 R: AAGGTGGTAGTTGCTCCTGAAGGT MMP1b F: AGTCTTTGAGGAGGAAGGCGATA MMP1b R: CAAACCTAGGCCTGGCAGAA MMP9 F: ACGACATAGACGGCATCCAGTATC MMP9 R: AGGTATAGTGGGACACATAGTGGG MMP19 F: GTGTGGACTGTAACAGATTCAGGG MMP19 R: CCTTGAAGAAATGAGTCCGTCGTG Birc2 F: TGATGGTGGCTTGAGATGTTGGGA Birc2 R: TGAATCTCATCAACAAACTCCTGACCC Birc3 F: TGTCAGCCAAGTTCAAGCTG Birc3 R: ATCTTCCGAACTTTCTCCAGGG Yap1 F: AAGGCCATGCTTTCGCAACT Yap1 R: ATCCTGAGTCATGGCTTGCT
2 Supplementary Figure Legends Figure S1. Southern blot hybridization results on tumor and tumor cell DNA confirms that the MMP13 and MMP7 genes are amplified. Genomic DNA cleaved with BamHI was subjected to agarose gel electrophoresis and blotted to nitrocellulose before hybridization to labeled MMP13, MMP7, and p53 probes indicated at right. Phosphorimager quantitation of band intensities relative to the p53 pseudogene control (***) are indicated under each band in the upper two panels. In the lower p53 panel, the upper most band (***) is the p53 pseudogene, the middle band (**) is the p53 null allele and the bottom band (*) is the p53 wildtype allele. The two left lanes labeled c2846 and c2984 contain DNA from two tumors shown to be positive for the chromosome 9A1 amplicon by BAC array CGH analysis. The two lanes to the right show DNA from cell lines derived from each of these two tumors. The central Ctrl lane contains DNA from normal diploid tail DNA. Figure S2. MMP family gene mrna expression as measured by RNA microarray analysis. The heat map shows relative expression levels of MMP family members separated into two groups: MMPs on the chromosome 9A1 amplicon (listed in the order of their relative locations on the chromosome) and MMPs outside of the chromosome 9A1 amplicon. Expression levels are relative to mc3t3 osteoblast cells (left most lane). Red indicates high expression relative to mc3t3 cells and green represents low expression (see logarithmic scale at upper right). OS: osteosarcoma. A+: chromosome 9A1 amplicon positive. A-: chromosome 9A1 amplicon negative. RS: rhabdomyosarcoma. Some genes have more than one row of data, which indicates that there is more than one probe for the same gene on the microarray chip. Figure S3. Expression of several MMP family genes in mouse and human osteosarcomas. Mouse: data is from real-time PCR. Sample number: 1-2: amplicon-positive (chromosome 9A1) osteosarcomas. 3-15: amplicon-negative
3 osteosarcomas : rhabdomyosarcomas. Human: data was obtained from RNA microarray analyses from 147 human osteosarcoma samples. MMP8, MMP12, and MMP20 were not available from the human data. Figure S4. Primary and transplanted allograft osteosarcomas have similar histopathology. Hematoxylin and eosin staining of tumor sections from (a) a primary osteosarcoma, (b) a secondary osteosarcoma formed after subcutaneous transplant of clonal osteosarcoma cells into nude mice, and (c) a primary rhabdomyosarcoma. Figure S5. One snapshot of the three-dimensional re-constitution of the CT scanning of allografted, MMP13 shrna-expressing (right arrow) or vectorexpressing (left arrow) tumors. Figure S6. Expression of baculoviral-iap repeat-containing (Birc) family members by RNA microarray. mc3t3: osteoblasts control. OS: osteosarcoma. A+: chromosome 9A1 amplicon positive. A-: chromosome 9A1 amplicon negative. RS: rhabdomyosarcoma. Figure S7. RNA expression of Yes-associated protein 1 (Yap1) gene by RNA microarray analysis. mc3t3: osteoblasts control. OS: osteosarcoma. A+: chromosome 9A1 amplicon positive. A-: chromosome 9A1 amplicon negative. RS: rhabdomyosarcoma. There are two probes for Yap1 on the microarray chip, as shown by two rows of data. Figure S8. Yap1 mrna knockdown in the allografted tumors. Real-time PCR analysis was used to examine Yap1 mrna level in the allografted tumors. It was found that both of the Yap1 shrna vectors (-1 and -2) have efficiently reduced Yap1 expression in either A+ or A- cells.
4 Table 1. Copy number gains and losses in p53+/- osteosarcomas and rhabdomyosarcomas
5 Supplementary Figure 1
6 Supplementary Figure 2
7 Supplementary Figure 3
8 Supplementary Figure 4
9 Supplementary Figure 5 vector MMP13 shrna
10 Supplementary Figure 6
11 Supplementary Figure 7
12 Supplementary Figure 8 A+ A v shyap1-1 shyap v shyap1-1 shyap1-2
(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-
1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish
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Supplemental Figure 1
Supplemental Figure 1 A S100A4: SFLGKRTDEAAFQKLMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGFPDK Overlap: SF G DE KLM LD N D VDFQEY VFL I M N FF G PD S100A2: SFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDR
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p.r623c p.p976l p.d2847fs p.t2671 p.d2847fs p.r2922w p.r2370h p.c1201y p.a868v p.s952* RING_C BP PHD Cbp HAT_KAT11
ARID2 p.r623c KMT2D p.v650fs p.p976l p.r2922w p.l1212r p.d1400h DNA binding RFX DNA binding Zinc finger KMT2C p.a51s p.d372v p.c1103* p.d2847fs p.t2671 p.d2847fs p.r4586h PHD/ RING DHHC/ PHD PHD FYR N
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