oligonucleotide-conjugated beads, 5 of which carry p53 REs (ConA, ConC, P21, PUMA, GADD45) and a negative control (WRNC), were incubated with nuclear extracts (NT: non-treated; Doxo: Doxorubicin-treated) for 60 minutes in the presence of variable amounts of either non-competing (NC) or competing (Comp) oligonucleotides and analyzed for p53 binding (see Materials and Methods for sequence of oligonucleotides used). A) The oligonucleotide set was incubated with decreasing amounts of non-competing oligonucleotide (shown in pmoles per reaction) and 1.75 micrograms of nuclear extracts from Doxo-treated cells. B) Treatment with decreasing amounts of a competing oligonu- cleotide and 1.75 micrograms of nuclear extract from Doxo- treated cells. NC oligonucleotides were not added in this experiment. Under these conditions ,1 pmole of competing oligonucleotide is needed to eliminate p53 binding. The relative binding intensity (value shown on vertical axes) was obtained for each oligonucleotide as discussed in Materials and Methods. Note breaks in the scale of vertical axes. Values shown are means for each bead type6SD (n = 3). Found at: doi:10.1371/journal.pgen.1000462.s003 (0.06 MB PDF) Figure S4 Concentration versus binding isotherm for nuclear extracts containing activated p53. A multiplex set of 6 oligonu- cleotide-conjugated beads, five carrying p53 REs (ConA, ConC, P21, PUMA, GADD45) and one a negative control (WRNC), was incubated with variable amounts of DOXO treated nuclear extracts in the presence of 150 pmoles of non-competing oligonucleotides for 60 minutes and analyzed for p53 binding. A) Normalized binding for each oligonucleotide is shown with varying amounts of nuclear extracts from Doxo-treated cells. The relative binding intensity (value shown on vertical axes) was obtained for each oligonucleotide as discussed in Materials and Methods. Values shown are means for each bead type6SD (n = 3). Nonlinear regression was used to determine BMax and Kd. A concentration of nuclear extract that was saturating for the p21 RE (1.75 ug) was chosen for the assay. An additional parameter investigated included the impact of bead quantity on relative binding. Bead quantity was varied from 2500 to 40,000 beads (representing up to 80 bead types), each bead represented at equal concentration. These experiments were carried out under varying protein concentrations. Using 1.75 ug protein in the hybridization, no effect of bead number on binding was observed. At lower protein concentrations (less than 1.2 ug protein) binding values were reduced for higher bead quantities in a linear, bead-quantity dependent manner. Thus protein and bead/probe number need to be optimized for a given system. Found at: doi:10.1371/journal.pgen.1000462.s004 (0.05 MB PDF) Figure S5 Detection of the interaction between ERa and its cognate REs. An oligonucleotide set representing 8 previously validated ERa REs (see supplementary materials for complete sequences) was assembled and tested for ERa binding. The panel included ConA and WRNC (from the p53 RE set). A) The ERa RE panel was treated with nuclear extracts from cells with no estrogen receptor (Lymph NT, lymphoblasts; Lymph Doxo, Doxorubicin treated; U2OS NT, untransfected U2OS cells) or pSG5-ERa-transfected U2OS cells (U2OS+pSG5-ERa). ER a interaction with ERE sequences was assessed by treating with anti- ERa antibody. Bar values are means for each bead type+/2SD (n = 3). The relative binding intensity (value shown on vertical axes) was obtained for each oligonucleotide as discussed in Materials and Methods. Nuclear extracts from cells that do not express ERa (lymphocytes, Dox-treated lymphocytes and U2OS cells) produced very low signal. In contrast, ERa binding to bona fide EREs was strongly detected following incubation with extracts Sequence Variation and p53 DNA Binding from an ERa over-expressing cell line (U2OS2pSG5-ERa). B) Experimental binding vs calculated PWM for ERa. Comparison of experimental ERa binding (relative binding, n = 3) vs calculated ERa binding values (PWM). EBAG9, EFP, and COX7RP probes contain ERa binding sites closely matching the ERE consensus and displayed the highest level of binding to ERa. TERT, pS2, LTF and KRT19 contain one or two changes from consensus and showed moderate ERa binding, while CTSD had the lowest binding signal. Values shown are means for each bead type6SD (n = 3). Within this small set of EREs, the binding was highly correlated (R2 = 0.78, p,0.001) with their sequence match to consensus (based on calculated PWM values). For instance, EBAG9, EFP, and COX7RP contain ERa binding sites closely matching the ERE consensus and displayed the highest level of binding to ERa. TERT, pS2, LTF and KRT19 contain one or two changes from consensus and showed moderate ERa binding, while CTSD had the lowest binding signal. Unexpectedly, ERa binding was observed for the p53 positive and negative control sequences ConA and WRNC. Further evaluation using an ERE PWM model revealed that these sequences contain ERE-like motifs that score as high as CTSD target sequences. This assay requires further optimization. Found at: doi:10.1371/journal.pgen.1000462.s005 (0.05 MB PDF) Figure S6 Multiplex capability of MAPD using extracts from untreated and treated cells. A total of 60 oligonucleotides, each bearing a single nucleotide variation of a perfect p53 consensus binding site (ConA = GGGCATGTCCGGGCATGTCC) was generated. ConA sequence is shown in large letters. The CATG core of each half-site is shown in a right-facing bracket. Systematic base substitutions at all positions within ConA are shown in small letters. For instance, 1G.C is an RE with the sequence CGGCATGTCCGGGCATGTCC. In addition, a positive con- trol bead (ConA RE) and a negative control bead (WRNC: lacks a p53 binding RE) were also generated. Each oligonucleotide variant was coated onto a unique microsphere type. Equal amounts of each microsphere type were mixed to generate a multiplex of 62 types of microspheres. Up to 82 oligonucleotides have been successfully multiplexed (not shown). Beads were incubated with nuclear extracts from untreated cells (NT, checkered bar) or nuclear extracts from doxo-activated cell (Doxo, black bars). Data from Doxo treated cells in this figure are also plotted in Figure 3b. The relative binding intensity (value shown on upper horizontal axis) was obtained for each oligonucleotide (white bar) as discussed in Materials and Methods. Bar values are means for each bead type6SD (n = 3). Found at: doi:10.1371/journal.pgen.1000462.s006 (0.67 MB PDF) Figure S7 Comparative binding. A) Normalized binding values from Doxo-induced p53 binding for p53 REs shown in Figure 6 are plotted against binding values for Wt p53 expressed in SaOS- 2 cells (data from Figure 6A). Binding values between the two extracts are highly correlated, linear regression line is plotted, r2 = 0.86. B) Normalized binding values (n = 8) from Doxo- induced p53 binding for p53 REs shown in Figure 4C are plotted against available ChIP-PET-SAGE values from Wei et al [31] downloaded from the UCSC browser. A correlation coefficient of r2 = 0.58 (p = 0.01) was observed for the comparison of MAPD- determined relative binding vs ChIP-PET-SAGE for 8 known p53 REs despite using different cell lines, treatments, and methods. Found at: doi:10.1371/journal.pgen.1000462.s007 (0.04 MB PDF) Figure S8 Evaluation of eight p53 RE SNPs using nuclear extracts containing activated p53 from DOXO treated U2OS cells (same procedure as Figure 5). PLoS Genetics | www.plosgenetics.org 11 May 2009 | Volume 5 | Issue 5 | e1000462