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Supplementary Data Supplementary Figure 1 Supplementary Figure 2 CCR-10-3244.R1 Supplementary Figure Legends Supplementary Figure 1. B-Myb is overexpressed in primary AML blasts and B-CLL cells. Baseline B-Myb mRNA levels were determined by quantitative RT-PCR, after normalization to the level of housekeeping gene, in primary B-CLL (n=10) and AML (n=5) patient samples, and in normal CD19+ (n=5) and CD34+ (n=4) cell preparations. Each sample was determined in triplicate. Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Supplementary Figure 2. Cytotoxicity by Nutlin-3 and Chlorambucil used alone or in combination in leukemic cells. The p53wild-type EHEB and SKW6.4 cells lines, and the p53mutated BJAB cell line were exposed to Nutlin-3 or Chlorambucil used either alone or in combination. (Nutl.+Chlor.). In A, upon treatment with Nutlin-3 or Chlorambucil, used either alone (both at 10 μM) or in combination (Nutl.+Chlor.), induction of apoptosis was quantitatively evaluated by Annexin V/PI staining, while E2F1 and pRb protein levels were analyzed by Western blot. Tubulin staining is shown as loading control. The average combination index (CI) values (analyzed by the method of Chou and Talalay) for effects of Chlorambucil+Nutlin-3 on cell viability are shown. ED indicates effect dose. In B, levels of B-Myb and E2F1 mRNA were analyzed by quantitative RT- PCR. Results are expressed as fold of B-Myb and E2F1 modulation in cells treated for 24 hours as indicated, with respect to the control untreated cultures set to 1 (hatched line). Data are reported as means+SD of results from at least three experiments, each performed in triplicate. Asterisks, p<0.05 with respect to the single treatment (A) or to the untreated cultures (B). CCR-10-3244.R1 Supplementary Material and Methods cDNA microarray For cDNA microarray, 3 µg of total RNA were transcribed into cDNA using GEArray AmpoLabeling-LPR Kit (Superarray Bioscience Corporation, Frederick, MD). Labeled cDNA was hybridized with a customized cDNA microarray (Oligo GEArray® Human Cancer Microarray OHS-802), containing 440 genes mainly associated to apoptosis, cell cycle, cell growth and differentiation, signal transduction together with housekeeping genes (Supplementary Table 1). Signal intensity was measured for each microarray, the minimal intensity was used for background subtraction and the values were normalized to the median signal value for each array. Expression levels were compared between the leukemic and the normal PBMC samples, then data were filtered for the genes whose expression level increased or decreased by at least two fold, that is, filtering the ratio for values ≥2.0 or ≤0.5. For microarray results validation and for analysis of B-Myb, E2F1, p53 and p21 gene expression, in either untreated or treated cultures, we have used the SYBR Green real-time PCR detection method, using the SABiosciences RT2 Real-TimeTM Gene Expression Assays, that include specific validated primer sets and PCR master mixes (SABiosciences, Frederick, MD). All samples were run in triplicate with the Real Time Thermal Analyzer Rotor- GeneTM 6000 (Corbett, Cambridge, UK). Expression values were normalized to the housekeeping gene POLR2A amplified in the same sample. SiRNA experiments For specific gene knock-down, siRNA were designed and manufactured by Ambion Inc. (Woodward Austin, TX) according to the current guidelines for effective gene knock-down by this method. Based on preliminary validation experiments, the following siRNA were selected: target#1 for p53, 5'-GGGAGUUGUCAAGUCUUGCtt-3' (sense) and 5'-GCAAGACUUGACAACUCCCtc- 3' (antisense); target#2 for p53, 5'-GGGUUAGUUUACAAUCAGCtt-3' (sense) and 5'- 2 CCR-10-3244.R1 GCUGAUUGUAAACUAACCCtt-3' (antisense); target#1 for E2F1, 5'- GGCCCGAUCGAUGUUUUCCtt-3' (sense) and 5'-GGAAAACAUCGAUCGGGCCtt-3' (antisense); target#2 for E2F1, 5'-CCUGAUGAAUAUCUGUACUtt-3' (sense) and 5'- AGUACAGAUAUUCAUCAGGtg-3' (antisense); target#1 for B-MYB/MYBL2, 5’- CCACAUCGAAGGAACAGGAtt-3’ (sense) and 5’-UCCUGUUCCUUCGAUGUGGtg-3’ (antisense); target#2 for B-Myb, 5’-CACUGGAACUCUACCAUCAtt-3’ (sense) and 5’- UGAUGGUAGAGUUCCAGUGat-3’ (antisense); target#3 for B-Myb, 5’- CCCUGUCAGGUAUCAAAGAtt-3’ (sense) and 5’-UCUUUGAUACCUGACAGGGtg-3’ (antisense). A cocktail of three different negative control siRNAs, each comprised of a 19 bp-scrambled sequence with 3’ dT overhangs (Ambion’s Silencer negative control siRNA), was used to demonstrate that the transfection did not induce non-specific effects on gene expression. The Ambion’s Silencer negative control siRNA sequences have no significant homology to any known gene sequences from human and they have been previously tested for the lack of non-specific effects on gene expression (Ambion). 3 Supplementary Table 1. OHS802 gene array Position GeneBank Symbol Description 1 NM_002954 RPS27A Ribosomal protein S27a 2 NM_002954 RPS27A Ribosomal protein S27a 3 NM_001605 AARS Alanyl-tRNA synthetase 4 NM_000927 ABCB1 ATP-binding cassette, sub-family B (MDR/TAP), member 1 5 NM_005845 ABCC4 ATP-binding cassette, sub-family C (CFTR/MRP), member 4 6 NM_005759 ABI2 Abl interactor 2 7 NM_005157 ABL1 V-abl Abelson murine leukemia viral oncogene homolog 1 V-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson- 8 NM_005158 ABL2 related gene) 9 NM_005781 TNK2 Tyrosine kinase, non-receptor, 2 10 NM_001610 ACP2 Acid phosphatase 2, lysosomal 11 NM_000666 ACY1 Aminoacylase 1 12 NM_000026 ADSL Adenylosuccinate lyase 13 NM_000476 AK1 Adenylate kinase 1 14 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 15 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 16 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 17 NM_002954 RPS27A Ribosomal protein S27a Aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; 18 NM_001354 AKR1C2 bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III) 19 NM_005163 AKT1 V-akt murine thymoma viral oncogene homolog 1 20 NM_000477 ALB Albumin Alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, 21 NM_001150 ANPEP microsomal aminopeptidase, CD13, p150) 22 NM_001154 ANXA5 Annexin A5 23 NM_004034 ANXA7 Annexin A7 24 NM_004068 AP2M1 Adaptor-related protein complex 2, mu 1 subunit 25 NM_000038 APC Adenomatosis polyposis coli 26 NM_001664 RHOA Ras homolog gene family, member A 27 NM_004040 RHOB Ras homolog gene family, member B 28 NM_175744 RHOC Ras homolog gene family, member C 29 NM_014578 RHOD Ras homolog gene family, member D 30 NM_001173 ARHGAP5 Rho GTPase activating protein 5 31 NM_005435 ARHGEF5 Rho guanine nucleotide exchange factor (GEF) 5 32 33 NM_002892 ARID4A AT rich interactive domain 4A (RBP1-like) 34 NM_183356 ASNS Asparagine synthetase 35 NM_001675 ATF4 Activating transcription factor 4 (tax-responsive enhancer element B67) Ataxia telangiectasia mutated (includes complementation groups A, C and 36 NM_000051 ATM D) ATP synthase, H+ transporting, mitochondrial F1 complex, beta 37 NM_001686 ATP5B polypeptide ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit 38 NM_001697 ATP5O (oligomycin sensitivity conferring protein) 39 NM_001699 AXL AXL receptor tyrosine kinase 40 NM_000465 BARD1 BRCA1 associated RING domain 1 41 NM_004324 BAX BCL2-associated X protein 42 NM_000633 BCL2 B-cell CLL/lymphoma 2 43 NM_003670 BHLHB2 Basic helix-loop-helix domain containing, class B, 2 44 NM_000386 BLMH Bleomycin hydrolase 45 NM_004333 BRAF V-raf murine sarcoma viral oncogene homolog B1 46 NM_007294 BRCA1 Breast cancer 1, early onset 47 NM_000059 BRCA2 Breast cancer 2, early onset 48 NM_000061 BTK Bruton agammaglobulinemia tyrosine kinase 49 NM_001746 CANX Calnexin 50 NM_006367 CAP1 CAP, adenylate cyclase-associated protein 1 (yeast) 51 NM_005186 CAPN1 Calpain 1, (mu/I) large subunit 52 NM_001749 CAPNS1 Calpain, small subunit 1 53 NM_001753 CAV1 Caveolin 1, caveolae protein, 22kDa 54 NM_001755 CBFB Core-binding factor, beta subunit 55 NM_170662 CBLB Cas-Br-M (murine) ecotropic retroviral transforming sequence b 56 NM_002982 CCL2 Chemokine (C-C motif) ligand 2 57 NM_053056 CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) 58 NM_001759 CCND2 Cyclin D2 59 NM_001760 CCND3 Cyclin D3 60 NM_001238 CCNE1 Cyclin E1 61 NM_012073 CCT5 Chaperonin containing TCP1, subunit 5 (epsilon) 62 NM_013230 CD24 CD24 antigen (small cell lung carcinoma cluster 4 antigen) 63 NM_000610 CD44 CD44 antigen (homing function and Indian blood group system) CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, 64 NM_000611 CD59 EJ16, EJ30, EL32 and G344) 65 NM_001255 CDC20 CDC20 cell division cycle 20 homolog (S. cerevisiae) 66 NM_001789 CDC25A Cell division cycle 25A 67 NM_004358 CDC25B Cell division cycle 25B 68 NM_001790 CDC25C Cell division cycle 25C 69 NM_003718 CDC2L5 Cell division cycle 2-like 5 (cholinesterase-related cell division controller) 70 NM_003674 CDK10 Cyclin-dependent kinase (CDC2-like) 10 71 NM_000075 CDK4 Cyclin-dependent kinase 4 72 NM_004935 CDK5 Cyclin-dependent kinase 5 73 NM_001261 CDK9 Cyclin-dependent kinase 9 (CDC2-related kinase) 74 NM_004196 CDKL1 Cyclin-dependent kinase-like 1 (CDC2-related kinase) 75 NM_000389 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 76 NM_004064 CDKN1B Cyclin-dependent kinase inhibitor 1B (p27, Kip1) 77 NM_000076 CDKN1C Cyclin-dependent kinase inhibitor 1C (p57, Kip2) 78 NM_058195 CDKN2A Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 79 NM_004936 CDKN2B Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)
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