Fig. 1
CDK12/13 inhibition results in selective cytotoxicity in NB cells and affects transcription elongation. a Dose-response curves for human NB cells treated with increasing concentrations of THZ531 for 72 h. Kelly E9R cells, which express a homozygous mutation at the Cys1039 THZ531-binding site in CDK1220 (see Methods) were also included. Fibroblast cells (NIH-3T3, IMR-90, BJ) were used as controls. Cytotoxicity is reported as percent cell viability relative to DMSO-treated cells. Data represent mean ± SD; n = 3. b Western blot analysis of Pol II phosphorylation in NB cells treated with THZ531 or DMSO at the indicated concentrations for the indicated times. c Waterfall plot of fold-change in gene expression in IMR-32 NB cells treated with THZ531, 400 nM for 6 h; selected DDR genes are highlighted. d qRT-PCR analysis of the indicated DDR gene expression in Kelly WT (left) cells and Kelly E9R (right), treated with THZ531 or DMSO at the indicated concentrations for 6 h. Data are normalized to GAPDH and compared to the DMSO control. e Flow cytometry analysis of γ-H2AX staining in Kelly NB cells treated with 400 nM THZ531 for the indicated time points (left). Gating was performed as shown in the left panel. Numbers indicate the percentages of living cells that stained positive for γ-H2AX. Quantification of staining (right). f Immunofluorescence staining of RAD51 focus formation in Kelly NB cells treated with THZ531 (400 nM) or DMSO for 24 h prior to exposure to gamma radiation (IR, 8 Gy). Nuclei are stained with DAPI (scale bar, 10 µM). Quantification of staining (right) of RAD51+ cells (>5 RAD51 foci per cell). Throughout the figure, error bars indicate mean values ± SD of three independent experiments, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test
