iHAP1

The depletion of Rod1 and TACC3 results in sensitization to iHAP1, which is caused by chemogenic interaction between Rod1 and iHAP1. As for the RPE1‐hTERT P53−/− background, deletion of Rod1 in HeLa cells causes marked sensitization to iHAP1.^[1]

The Sulforhodamine B (SRB) proliferation assay was used to assess in vitro cytotoxicity of the compounds. Cells were treated with sublethal drug concentrations (LD20) for 12 days from the day of seeding. If necessary, transient siRNA transfection was performed 3 days before drug treatment. After 12 days, medium was removed from the wells and cells were washed once with PBS and fixed in prechilled 10% trichloroacetic acid (TCA) for 30 min at 4°C. Cells were subsequently washed two times with double‐distilled water and stained with 0.4% SRB (1% acetic acid for 20 min at room temperature protected from light). SRB was then removed, and cells were washed four times in 1% acetic acid. After complete light‐protected drying, SRB was dissolved by addition of 10 mM Tris pH 8 and gentle shaking at room temperature for 2 h. The absorbance at A510 was read. Percentage of cell growth inhibition was calculated. The IncuCyte® live‐cell analysis system was additionally used to real‐time track the growth of the cells subjected to the different treatments.