mock treated control, Wortmannin manufacturer belinostat and VPA treatment in HeLa cells. The doses chosen were close to the IC50 values in HeLa 0. 76 M and 3. 3 mM for belinostat and VPA respectively, and induced histone H3 and H4 hyper acetylation. Differential gene expression patterns were detected between each experimental condition versus control. Fig. 3 summarizes the number of non redundant genes significantly deregulated in response to drug treat ment, at an arbitrary 2. 0 fold change cut off value. The number of genes deregulated by belinostat or VPA is 5. 3 and 6. 0% respectively. Further, a greater proportion of genes are induced by both drugs. The relationship of differentially expressed genes between belinostat and VPA was illustrated by Venn dia grams, and demonstrated that approximately 30% of altered genes responded identically between drug treat ments.

In the literature, certain genes have been identified whose expression is affected by HDACi treatment. For instance, Glaser et al. recognized a common set of 13 core genes whose expression were universally altered in response to various HDACi in multiple cell types. Some of the commonly affected genes are listed in Table 1 for belinostat and VPA treatment, and these include up regulation of CDKN1A and FUCA and down regu lation of TYMS, CTPS and KPNB1 by both drugs. Hence, this study confirms a subset of 5 of the 13 core genes besides other known HDACi target genes. Comprehensive gene lists of all conditions at above 2 fold changes are accessible.

Data were validated by qRT PCR analysis on 9 selected genes on RNA samples used in microarray analysis plus inde pendent ones, and an overall good correlation to the microarray data was observed. Gene expression profiles of class I HDAC depletion Further, we conducted a genome wide analysis of the tran scriptional response to siRNA mediated depletion of three class I HDACs in HeLa cells. As for HDACi samples, DNA chip analyses were carried out in independent triplicates for each condition. scrambled siRNA control, HDAC1, 2 and 3 KD at 48 hours post transfection. Differential gene expression patterns between each knockdown condition and scrambled control were identified by statistical analy sis. Efficient KD was confirmed by the microarray data, as each HDAC isoform was specifically down regulated 7 10 fold.

The proportion of non redundant significant transcripts affected by the down regulation of each HDAC enzyme at 2. 0 fold change or more is in the range 1. 4 2. 0%, and highest for HDAC1 KD. As for HDACi drugs, a slight overweight of transcripts was induced AV-951 for HDAC1 and 2 KD samples. In contrast, HDAC3 KD was the only condition not showing this pat tern. The proportion of genes with identical expression between KD conditions was in the order of 19 27%, with HDAC1 KD displaying the least overlap with the other two KD conditions.