Fig. 5: Effect of the TOP2A inhibitor etoposide on the PRRX1A–TOP2A interaction.

a Structure of TOP2A in the presence of etoposide and ATP (6ZY8). To allow the binding site of TOP2A to be seen, the DNA-gate and C-gate of TOP2A are translucent. b At 6ZY8, the width of the narrowest PRRX1A binding site was measured by PyMOL. Amino acid residues in the vicinity of the measurement site were plotted using a space-filling model (PRRX1A: residues 100:170, TOP2A: residues 350:360). The line segments used for the measurements are indicated by red dotted lines. c Inhibition of the PRRX1A–TOP2A interaction by etoposide. After treating HS-PSS/3xFLAG-PRRX1A/3xHA-TOP2A cells with 1 µg/mL DOX and DMSO (vehicle) or each indicated concentration of etoposide, cell lysates were extracted to immunoprecipitate 3xFLAG-PRRX1A. Input and each immunoprecipitated sample were detected with anti-FLAG or anti-HA antibody. d Semiquantitative analysis of HA-TOP2A levels. The western blot band intensities were measured using ImageJ software. HA-TOP2A protein levels were normalised to FLAG-PRRX1A levels. The intensity (mean ± standard error) was normalised to control values. Statistical significance was determined using unpaired one-way ANOVA with Tukey’s post hoc analysis. e Schematic illustration of the proposed mechanism by which the PRRX1-TOP2A interaction is inhibited by etoposide. The action of etoposide on TOP2A inhibits binding to PRRX1. Inhibition of the interaction between PRRX1 and TOP2A by etoposide stops the progression of DNA cleavage, causing double-strand DNA breaks and etoposide exerts its anticancer effect. Data are presented as the means ± SEMs. * p < 0.05; ** p < 0.01.