(A–C) Comparison of the number of cells with 53BP1-Apple-trunc fluorescence above the baseline in U251MG WTE and TLS Polκ KO 3D tumor spheroids evaluated after 24- (A), 48- (B), and 72-h (C) treatments with TMZ and their respective controls. (D,E) Photomicrographs of 3D tumor spheroids of U251MG WTE (D) and TLS Polκ KO (E) cells obtained after treatment 0, 24, 48, and 72 h with TMZ and respective controls. All images were obtained by the EVOS XL core microscope using an RFP (red) channel and 10× objective. The images were analyzed and quantified by the software Fiji v. 3.1. Scale: 400 μm (white bar). All data were represented as means ± standard deviation (X ± SD) from four spheroids (n=4) per replicate and three independent biological experiments (n=3). *Values statistically different from the WTE cells at the point (date) (*P<0.05, **P<0.01, ***P<0.001; Two-way ANOVA followed by Bonferroni post-test). PC: Positive Control (Cisplatin 100 μM); SC: Solvent Control (1% DMSO); TMZ: Temozolomide.