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1 Supporting Information Vaira et al /pnas h 24h 48h PTEN pathway Expression 0h 24h 48h Time MAP Kinase Signaling Pathway Expression 0h 24h 48h Time Collagen Expression Fig. S1. Differential gene-expression analysis of tumor and stroma samples over time. The plots depict, with a blue line, the loess-smoothed expression profiles of three gene sets: PTEN (phosphatase and tensin homolog deleted on chromosome 10) pathway, MAP kinase signaling pathway and collagen, and distinctive of tumor (PTEN and MAPK pathway) and stromal (collagen) compartments, respectively. The standard error gray band and cross-sample median line are shownfor perspective. For each of these gene sets, the distinction between the mean profiles of tumor and stroma classes was found to be statistically significant. Time A B DMSO Nutlin-3 10 % BrdU DMSO Nutlin-3 p21 mdm2 Fig. S2. Mdm2 inhibition in dedifferentiated liposarcoma. A shows a reduction in proliferation, assessed by BrdU incorporation, in tumors treated with the Mdm2 inhibitor Nutlin-3 compared with DMSO after 24 h of culture. Immunostaining pictures of p21 and Mdm2 protein expression are presented in B. 1of6
2 Table S1. Immunohistochemical data for individual tumors (untreated) Tissue Sample ID Time (h) Ki-67% p-akt p-s6rp Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon x x Colon Colon Colon Colon Colon Colon Lung Lung Lung Lung Lung x Lung x Lung x Lung x Lung x Lung x Lung Lung x x Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung x x Lung Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate X, data not available. 2of6
3 Table S2. TUNEL assay for individual tumors Tissue Sample ID Time (h) TUNEL (%) Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Prostate Prostate Prostate Prostate Prostate Prostate Prostate of6
4 Table S3. Organ-specific molecular analysis: Evaluation of Akt/PI3K pathway in colon (T = 7;n = 2) Timepoints (h) PI3K AKT1 p70-s6k FAK-PTK2 Tumor ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.45 Normal ± ± ± ± Results are expressed as averaged relative quantities of indicated targets ± SEM. Table S4. Organ-specific molecular analysis: Evaluation of Akt/PI3K pathway in lung (T = 6; n = 3) Timepoints (h) PIK3 AKT1 p70-s6k FAK-PTK2 Tumor ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.27 Normal ± ± ± ± ± ± ± ± Results are expressed as averaged relative quantities of indicated targets ± SEM. Table S5. Organ-specific molecular analysis: Evaluation of Akt/PI3K pathway in prostate (T =3;n =2) Timepoints (h) PI3K AKT1 p70-s6k FAK-PTK2 Tumor ± ± ± ± ± ± ± ± Normal ± ± ± ± Results are expressed as averaged relative quantities of indicated targets ± SEM. Table S6. Evaluation of Ki-67 and MTT after treatment with the PI3K inhibitor LY Timepoints (h) Ki-67 (DMSO) Ki-67 (LY) MTT (DMSO) MTT (LY) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05 Results are expressed as averaged values ± SEM relative to baseline (T0). 4of6
5 Table S7. Gene expression of Akt/PI3K pathway target on treatment of tumor slices (n = 9) with PI3K inhibitor LY Timepoints (h) FAK-PTK2 AKT1 p70-s6k DMSO ± ± ± ± ± ± ± ± ± 0.05 LY ± ± ± ± ± ± ± ± ± 0.05 Results are expressed as averaged relative quantities of indicated targets ± SEM. Table S8. tumors Proliferative and apoptotic markers at increasing doses of LY in individual Tissue Sample ID Time (h) LY Dose (μm) Ki-67 (%) TUNEL (%) Colon Colon Colon Colon Colon Colon Colon Colon Colon Colon Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung of6
6 Table S9. List of normal and pathological samples used in the study Organ (n) Diagnosis Pathological sample (n = 213) Breast (52) Carcinoma 52 Bladder (3) Carcinoma 3 Colon (53)* Adenoma 3 Adenocarcinoma 49 Other (nontumoral lesion) 1 Soft tissue (3) Liposarcoma 3 Liver (6)* Hepatocellular carcinoma 1 Hepatocellular Adenoma 1 Other (metastasis from Ca colon/lung) 4 Lung (32)* Non small-cell lung cancer 30 Other (nontumoral lesion, metastasis, mesothelioma) 2 Kidney (10)* Carcinoma 10 Ovary (4) Carcinoma 4 Pancreas (3)* Carcinoma 3 Prostate (27)* Carcinoma 27 Stomach (14) Carcinoma 10 GIST 3 other (nontumoral lesion, metastasis) 1 Uterus (4)* Carcinoma 4 Lymph node (2) Non-Hodgkin lymphoma 2 *For these organs, 58 normal samples were also analyzed: colon (8), liver (3), lung (9), kidney (8), pancreas (2), prostate (27), and uterus (1). Table S10. List of assay-on-demand (AB) used in qpcr experiments Target Assay-on-demand code Notes PI3K Hs _m1 AKT1 Hs _m1 p70-s6k Hs _m1 FAK-PTK2 Hs _m1 HMBS Hs _m1 housekeeping gene UBC Hs _m1 housekeeping gene ACTB Hs _m1 housekeeping gene 6of6
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