Supplementary Figure 5 from Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells

Figure S5. (A) PM030779 did not induce RNA Pol II degradation and DNA breaks. For the kinetics of RNA synthesis A549 cells were treated with the compound or DMSO in normal growth medium for 0, 30, 45, 60, 90 and 120 minutes and pulsed with 5µCi/ml [3H] uridine for 30min. Data are mean{plus minus}SEM values (triplicates, n=1). We have also evaluated Pol IIo and Pol IIa by western blot after treatment of A549 cells with PM030779 (30nM) for different time intervals. Finally, we performed Tail Moment quantification of A549 cells treated with PM030779 at 4h using the TriTek CometScore Freeware software. The Comet assay was performed in neutral conditions to detect double strand breaks (DSBs). For each condition a minimum of 100 cells were analysed, and the experiment was repeated in duplicate. (B) RNA synthesis inhibition, Pol II degradation and DNA breaks formation after treatment of OGROV-1 and IGROV-ET cancer cells with lurbinectedin For the kinetics of RNA synthesis both cell lines were treated with lurbinectedin (30nM) or DMSO in normal growth medium for 0, 30, 45, 60, 90 and 120 minutes and pulsed with 5µCi/ml [3H] uridine for 30min. Data are mean{plus minus}SEM values (triplicates, n=1). We have also evaluated Pol IIo and Pol IIa by western blot in both cell lines after treatment with lurbinectedin (Lur) (30nM) for different time intervals. Finally, we performed Tail Moment quantification of cells treated with Lur (30nM) at 4h using the TriTek CometScore Freeware software. For each condition a minimum of 100 cells were analysed, and the experiment was repeated in duplicate.