Figure 6.

Transfection of TDP1 into the H4 cell line to elevate the TDP1/TOP1 activity ratio and increase the irinotecan IC₅₀. (A) TDP1 and TOP1 protein levels after transfection of H4 with wild type (WT-TDP1) or mutant (H263A-TDP1) clones. Cells were collected 24 h after transfection and 10 µg of WCEs were analyzed by western blots. Whole blots with molecular weight markers are shown in Figure S6. The signal from actin was used to control the loading of WCE proteins. Normalized intensity ratios are given for each band, with the intensity ratio of mock-treated control set as 1. The bar graphs show the average and standard deviation of measurements as error bars from all technical replicates (n = 3 or more); (B) TDP1 and TOP1 activity levels in H4 WCE from the transfection of WT-TDP1. Cells were collected 24 h post-transfection and 4 µg of WCE was used in the gel-based assay for TDP1 activity; (C) Irinotecan IC₅₀ for H4 cells transfected with the WT-TDP1 clone versus H263A mutant TDP1 clone (n = 7).