a b Exon 1β 1α 23 ARF p16INK4a MDM2 Cyclin D CDK4/6 p53 pRB TaqMan Probes ARF 5’ Primer p16INK4a 5’ Primer Exon 1/2 Common Primer Exon 2/3 Common Primers Defective p53 Intact p53 A. Melanoma cell lines clustered by expression of p53 targets and INK4a/ARF expression. The p53 response to ionizing radiation is indicated. Top cluster: Cells with intact p53 response show increased expression of the eight indicated p53 targets, and decreased expression of ARF. Bottom cluster: TaqMan and microarray results for the p16INK4- and ARF- specific transcripts. Cell lines that demonstrated increased p16INK4a expression either harbored RB deletion, p16INK4a point 2 mutation or CDK4 point mutation (Supp Table 1). Pair-wise variances (r ) for the log2 transformed CDKN2a microarray 2 2 2 results versus the log10 transformed TaqMan results are: vs. Exon 2/3 r =0.78; vs. Exon1α/2 r =0.32; vs. Exon 1β/2 r =0.38. Note all four WT lines (PMWK, Mel505, SKMEL187 and RPMI8322) lack evidence of p53 function by all assays. B. The INK4a/ARF locus and with TaqMan strategies. The ARF transcript originates from exon 1β while the p16INK4a transcript originates from exon 1α. Both transcripts splice to exon 2 but in alternate reading frames. ARF stabilizes p53 by inhibiting MDM2, while p16INK4a activates RB by inhibiting CDK4. Primers and TaqMan probes are shown for the real-time RT-PCR strategy. Shields et al., Supp Figure 1 SKMEL 28 U01 24h SKMEL WM2664 U01 48h WM2664 U01 24h 24 U01 48h SKMEL 24 U01 24h SKMEL 24 Untreated SKMEL 24 DMSO 48h SKMEL 24 DMSO 24h SKMEL WM2664 DMSO 24h WM2664 DMSO 48h WM2644 Untreated 28 Untreated SKMEL 28 DMSO 48h SKMEL 28 DMSO 24h SKMEL -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 relative to median expression Genes decreased by UO1 (863) HIF1A Hypoxia-inducible factor 1, alpha subunit NM_001530 RBBP8 Retinoblastoma binding protein 8 NM_002894 Homo sapiens, clone IMAGE:4337652, mRNA BC018676 EIF4EBP1 Eukaryotic translation initiation factor 4E binding protein EXOSC8 Exosome component 8 NM_181503 ENST00000321524 MCM7 MCM7 minichromosome maintenance deficient 7 (S. cerevisiae) c-MYC DIAPH3 Diaphanous homolog 3 (Drosophila) AK092024 SLBP Stem-loop (histone) binding protein NM_006527 CD200 CD200 antigen BC022522 2016247 DUSP6 Dual specificity phosphatase 6 NM_001946 SPRY2 Sprouty homolog 2 (Drosophila) NM_005842 Ris1 DHFR Dihydrofolate reductase BC003584 LOC221710 Hypothetical protein LOC221710 AK058186 DIAPH1 Diaphanous homolog 1 (Drosophila) NM_005219 PUSL1 Pseudouridylate synthase-like 1 NM_153339 MAFF V-maf fibrosarcoma oncogene NM_012323 ENST00000309608 RPL39L Ribosomal protein L39-like NM_052969 THC1577090 FLJ23186 Hypothetical protein FLJ23186 NM_024616 AW082201 LIM protein AK027217 NRG1 Neuregulin 1 NM_013956 TWIST1 Twist homolog 1 NM_000474 KRT15 Keratin 15 NM_002275 MT1E Metallothionein 1E (functional) NM_175617 ATF3 MT2A Metallothionein 2A NM_005953 MT2A Metallothionein 2A BC007034 MT2A Metallothionein 2A NM_005953 Genes increased by UO1 (607) FosL2/Fra2 LEF1 Lymphoid enhancer-binding factor 1 NM_016269 CGI-121 CGI-121 protein NM_016058 MGC14560 Protein x 0004 AF117229 PSMD14 Transmembrane anchor protein 1 NM_005805 PAICS Phosphoribosylaminoimidazole carboxylase c-Jun LOC253981 Hypothetical protein LOC253981 AK025431 PPIL1 Peptidylprolyl isomerase (cyclophilin)-like 1 NM_016059 PCNA Proliferating cell nuclear antigen NM_002592 SFRS7 Splicing factor, arginine/serine-rich 7, 35kDa L22253 MYOHD1 Myosin head domain containing 1 NM_025109 MCM3 MCM3 minichromosome maintenance deficient 3 NM_002388 CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) NM_053056 CDC6 CDC6 cell division cycle 6 homolog (S. cerevisiae) NM_001254 Shields et al., Supp. Figure 2: Supervised analysis of gene expression in three different B-RAF mutant cell lines with high baseline levels of pERK +/- treatment with UO126, a MEK inhibitor. SAM identifies 1470 genes (FDR=1.4%) that significantly correlate with UO126 treatment at two different time points (24 and 48 h) and that change in expression by more than two-fold. Selected genes and clusters are shown; the full list of genes is included in Supp. Table 4. Supp. Table 1: Mutational status, source and media for melanoma cell lines a Cell Line Source Base Media p16 ARF p16-CDK4-RB ARF-p53 N-RAS B-RAF p-ERK PMWK J. Arbiser MEM, 0.5X AAb High-?? High-p53 inact. Unknown p16-RB lesion p53 inact. WT WT Low Mel 505 J. Hansson RPMI 1640 Undetectable High-p53 inact. Undetectable p16 mRNA p53 inact. WT WT Low SKMEL187 A. Houghton DMEM Undetectable Undetectable HD of 9p21 p53 mut (Arg273His) WT WT Low RPMI8332 J. Hansson RPMI 1640 Low-?? High-p53 inact. Unknown p16-RB lesion p53 inact. WT WT Low SKMEL23 A. Houghton DMEM n.d. n.d. n.d. n.d. WT WT n.d. SKMEL173 A. Houghton DMEM Undetectable Undetectable HD of 9p21 HD of 9p21, p53 funct. Q61K WT High VMM39 C. Slingluff DMEM Undetectable High-p53 inact. Undetectable p16 mRNA p53 inact. Q61K WT Low Mel 224 J. Hansson RPMI 1640 Undetectable High-p53 inact. Undetectable p16 mRNA p53 inact. Q61R WT Med SKMEL103 A. Houghton DMEM Undetectable Undetectable HD of 9p21 HD of 9p21, p53 funct. Q61R WT Med SKMEL147 A. Houghton DMEM High-p16 pt mut. Undetectable p16 mut (Pro114Leu) HD of ARF, p53 funct.l Q61R WT Med SKMEL63 A. Houghton DMEM High-p16 pt mut. High-?? p16 mut (Arg112Pro) Unknown ARF -p53 lesion Q61K WT High SKMEL119 A. Houghton DMEM Undetectable Undetectable HD of 9p21 HD of 9p21, p53 funct. Q61R WT High UACC 257 M. Soengas RPMI 1640 High-??CDK4 mut. High-?? ??CDK4 mut. Unknown ARF -p53 lesion WT V600E High A375 J. Arbiser DMEM Undetectable Undetectable Undetectable p16 mRNA HD del of ARF, p53 WT & funct. WT V600E High WM2664 J. Arbiser DMEM Undetectable Undetectable HD of 9p21 HD of 9p21, p53 funct. WT V600D High A2058 J. Arbiser DMEM High-Rb Null Undetectable Rb null Undetectable ARF mRNA, p53 functl. WT V600E Med SKMEL24 ATCC MEM, 0.5X AAb Undetectable Undetectable HD of 9p21 HD of 9p21, p53 funct. WT V600E High SKMEL28 ATCC MEM, 0.5X AAb High-CDK mut. High-p53 inact. CDK4 Arg24Cys mut. p53 Mut (Leu145Arg) WT V600E High a All base media contain 10% FCS + pen/strep b 0.5X Non-essential Amino Acids (AA) HD = Homozygous Deletion Shields et al., Supp. Table 3 -- Significant Genes List -- SAM analysis by RAS v RAF v WT status Input Parameters Imputation Engine 10-Nearest Neighbor Imputer Data Type Multiclass Response Number of Permutations 100 Blocked Permutation? FALSE RNG Seed 1234567 Delta 0.45483 (Upper Cutoff, Lower Cutoff) (1.51806, -∞) Computed Quantities Computed Exchangeability Factor S0 0.082034176 S0 percentile 0 False Significant Number (Median, 90 percentile) (25.20675, 48.86231) False Discovery Rate (Median, 90 percentile) (4.93283, 9.56210) Pi0Hat 0.77559 511 Positive Significant Genes Row Gene:AGI HUM1 OLIGO A: Gene ID Score(d) Numerator(r) Denominator(s+s0) q-value (%) 11371 AGI_HUM1_OLIGO_A_23_P96383 SRPX || sushi-repeat-containing protein, X-linked || NM_006307 || 5.115464897 3.780608669 0.739054758 1.384986015 7872 AGI_HUM1_OLIGO_A_23_P399255 MGC33993 || hypothetical protein MGC33993 || NM_152737 || 5.030548127 2.990170346 0.594402493 1.384986015 19899 AGI_HUM1_OLIGO_A_32_P191441 FLJ32938 || hypothetical protein FLJ32938 || AF435960 || 3.923772435 2.05718328 0.524287102 1.384986015 13453 AGI_HUM1_OLIGO_A_24_P233995 FLJ22390 || hypothetical protein FLJ22390 || AK000035 || 3.88594523 1.487583653 0.382811276 1.384986015 14633 AGI_HUM1_OLIGO_A_24_P318656 ITGB3 || integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) || M35999 || 3.695059959 2.619169537 0.708830051 1.384986015 18848 AGI_HUM1_OLIGO_A_32_P117453 || || A_32_BS117453 || 2.78540883 1.553178661 0.557612457 1.384986015 1646 AGI_HUM1_OLIGO_A_23_P132115 SNF1LK || SNF1-like kinase || NM_173354 || 2.698734598 1.496496357 0.554517794 1.384986015 8687 AGI_HUM1_OLIGO_A_23_P452 DDR2 || discoidin domain receptor family, member 2 || NM_006182 || 2.672100991 1.315304681 0.492236141 1.384986015 6217 AGI_HUM1_OLIGO_A_23_P316460 MGC3036 || hypothetical protein MGC3036 || NM_138434 || 2.662934233 2.186052033 0.820918521 1.384986015 10168 AGI_HUM1_OLIGO_A_23_P71946 BSPRY || B-box and SPRY domain containing || NM_017688 || 2.660592474 1.68156749 0.632027455 1.384986015 8321 AGI_HUM1_OLIGO_A_23_P421513 LOC220594 || TL132 protein || NM_145809 || 2.621822577 0.949302261 0.362077232 1.384986015 18039 AGI_HUM1_OLIGO_A_24_P918147 FBXO32 || F-box only protein 32 || AK023391 || 2.589683623 2.578443227 0.995659549 1.384986015 9177 AGI_HUM1_OLIGO_A_23_P52589 C11orf13 || chromosome 11 open reading frame 13 || NM_003475 || 2.583426595 1.16540778 0.451109307 1.384986015 10379 AGI_HUM1_OLIGO_A_23_P7642 SPARC || secreted protein, acidic, cysteine-rich (osteonectin) || NM_003118 || 2.578367061 3.487454305 1.352582554 1.384986015 9095 AGI_HUM1_OLIGO_A_23_P50919 SERPINE2 || serine (or cysteine) proteinase inhibitor, clade E || NM_006216 || 2.558234383 2.630409077 1.028212698 1.384986015 20282 AGI_HUM1_OLIGO_A_32_P214860 || || THC1424520 || 2.548945491 1.231610608 0.483184365 1.384986015 3092 AGI_HUM1_OLIGO_A_23_P159671 PHKA2 || phosphorylase kinase, alpha 2 (liver) || NM_000292 || 2.532558642 0.85362682 0.337061028 1.384986015 11030 AGI_HUM1_OLIGO_A_23_P89062 MGC3020 || hypothetical protein MGC3020 || NM_024048 || 2.510583793 0.773541968 0.308112388 1.384986015 19486 AGI_HUM1_OLIGO_A_32_P161836 || || I_1110147 || 2.50312346 0.70515112 0.281708486 1.384986015 13815 AGI_HUM1_OLIGO_A_24_P257348 JWA || cytoskeleton related vitamin A responsive protein || NM_006407 || 2.502422096 1.17564798 0.469804028 1.384986015 4410 AGI_HUM1_OLIGO_A_23_P209499