Figure 3

Microarray analysis validation and functional network for ΔAP-2γ/EGFP- expressing N202.1A breast cancer cells. (A) Chromatin from N202.1a cells was cross-linked to proteins, extracted and immunoprecipitated with either AP-2α (H-79) or AP-2γ (6E/4) Abs or non-specific rabbit- or mouse-IgG (negative isotype controls) or H3-histone or RNA-polymerase II Abs (positive controls). DNA was analyzed by PCR, using primers flanking the AP-2 putative binding sites in Ctgf and Tnfaip3 promoters. Input: non immunoprecipitated DNA. (B) Microarray data (additional file 1: Table S1 and Table 1) were validated by qRT-PCR performed in triplicate for 9 genes on three different RNA preparations from ΔAP-2γ/EGFP- (Δ#7; Δ#15) or EGFP- (Co#5; Co#11) expressing N202.1A clones. Black bars: microarray results; Dark grey bars: qRT-PCR of Δ#7 and Δ#15 clones versus Co#5 and Co#11 clones; Light grey bars: qRT-PCR Δ#7 clone versus Co#11 clone. The GAPDH gene was used as an internal control to normalize the data. Microarray analysis and qRT-PCR-fold changes are shown for each validated gene as average values. Bars represent ± standard error. (C) Functional network which connects the identified AP-2-regulated genes involved in "cell death" taken in part from analyses carried on with Ingenuity™ Pathway Analysis. Legend: Continuous grey lines indicate direct interactions experimentally proven; dashed grey lines represent potential indirect connections; dashed black lines represent potential indirect connections obtained from our microarray results considering only Fold Changes > 2. The green and red symbols represent down- and up-regulations, respectively, while the white symbols indicate genes absent in the dataset but related with the microarray genes as indicated from the literature. Blue checkmarks indicate the genes verified by qRT-PCR in (B).