Figure 4.

Dependence of CPT effects on antisense transcript levels on ongoing transcription. (A) The effects of 10 μM CPT for 4 h on antisense transcription have been studied after silencing of CDK9 (white bars) in HCT116 cells. Drug treatment was performed 72 h post-transfection of siRNAs. Scrambled siRNA samples are black bars. (B) CPT effects in HCT116 cells were studied at the selected locus with and without DRB. Co-treatment: cells were pretreated for 15 min with 50 μM DRB before the addition of CPT (10 μM) for 4 h. In all cases, PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means ± SEM of at least four determinations of two independent experiments for each panel. (C) Genomic map of the region −4000 to +1000 bp around transcription start site (TSS, bold line) of two analyzed genes having increased antisense transcripts induced by CPT. In each case, the top and bottom graphs reports the read distributions in HCT116 and HCT116-shRNATop1 cells, respectively. In shades of blue the patterns of sense reads, in shades of red those of antisense reads (dark colors for control cells; light colors for CPT-treated cells). In the centre of each graph, the map from UCSC genomic browser, highlighting the chromosomal position of unknown cluster, PCR amplicon, CpG island and the gene 5′-end, respectively. The validation by qRT-PCR has been conducted or in a region overlapping the unknown cluster (i.e. ATF1) or in a position close to it (i.e. GPC1), where it is evident an increase of antisense transcription simply by reads mapping.