Fig 1: The BLRR assay reveals HDR-suppressing effects of cardiac glycosides in GSC and GBM cells. (A) Schematic diagram of the BLRR assay in patient-derived GSCs. GSCs from patients were stably transfected with BLRR, then sequentially transfected with lentiviruses encoding trGluc and I-SceI, followed by treatment with small-molecule candidate HDR inhibitors to assess DSB repair by BLRR assay. (B) The BLRR assay reveals dose-independent HDR inhibition by HDR inhibitor CAY since the Gluc signal decreases as the amount of CAY is increased, while the Vluc signal remains stable. (C) The BLRR ratio demonstrates HDR inhibition in GSCs by CAY. (D, E) Both the BLRR signal (D) and the BLRR ratio (E) reveal dose-dependent inhibition of HDR by cardiac glycosides in U87-MG GBM cells. Lanatoside C, ouabain and digoxin were treated at the indicated concentrations. (F, G) Both the BLRR signal (F) and the BLRR ratio (G) reveal dose-dependent HDR inhibition in GSCs treated with lanatoside C, ouabain and digoxin. BLRR signals were normalised against cell viability and results are shown as the fold change relative to DMSO-treated controls (mean ± SEM of three biological replicates). The BLRR ratio is shown as the fold change relative to DMSO (mean ± SEM of three biological replicates). (H) Western blot analysis revealing cardiac glycoside-induced downregulation of RAD51 in U87-MG, as well as in GSCs (I). RAD51 protein levels in U87-MG and GSCs treated with the indicated cardiac glycosides at 250 and 1000 nM for 24 h. Significance was calculated using one-way ANOVA as indicated, followed by Tukey's post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).