Figure 6.

CPT determines a rapid and transient burst of Top1ccs that correlates with transient RNA Pol II block. (A) ICE bioassay was performed on HCT116 cells after 2, 5 and 20 min of 10 μM CPT treatment. The gel shows both CsCl gradient fractions containing DNA bound proteins (on the left) and free proteins (on the right). β-Actin was used as loading control. (B) ChIP on RNA Pol II. HCT116 cells were treated for 5, 10, 20 and 30 min with CPT 10 μM and the DNA recovery at indicated gene promoters is plotted as fold increase on not treated cells against treatment time. The analyzed gene regions are on TSS or immediately downstream to it and are listed (C). (C) List of genes observed in ChIP and K⁺SDS Assays [(B) and (D) panels], with the relative FPKM obtained by RNA-Seq. Used primers are reported in Supplementary Table SVI. (D) K⁺SDS precipitation assay on HCT116 and HCT116-shRNATop1 cells. Cells were treated for 2, 5, 10 and 20 min with 10 μM CPT. The recovery of Top1ccs at indicated gene promoters is reported as fold increase or percentage on input on not treated cells against treatment time. Used primers are in Supplementary Table SVI. (E) K⁺SDS precipitation assay on HCT116 in different positions along MYC and HIF-1α genes. Below the DNA recovery, a map showing the analyzed region. Standard errors (SEM) are not reported in (B) and (D) for image clarity. The average SEMs are as follows: 37% of the mean value (panel B); 19 and 37% of mean values for top and lower graphs (panel D HCT116), respectively; 16 and 23% of mean values for top and lower graphs (panel D HCT116-shRNATop1), respectively.