Figure 1. LDC526 is a potent CDK9 inhibitor inducing apoptosis of the MEC-1 cell line.

(A) Molecular structure of LDC526. (B) Analysis of CDK family selectivity of LDC526 in comparison to other CDK inhibitors. Red: IC₅₀ <0.1 μM; yellow: IC₅₀ ≥0.1 and <1μM, green: IC₅₀ ≥1 μM. (C) High CDK9 specificity of LDC526 in a panel of 219 kinases. (D) Rapid induction of apoptosis by LDC526. Apoptosis was assessed by Annexin V and DAPI staining after 4 hours of LDC526 incubation. Representative plots are shown. (E) Quantification of apoptotic cells (Annexin V+, DAPI-; n=3 independent replicates; incubation for 4 hours). (F) Intracellular flow cytometric analysis of MCL-1 and BCL-2 expression within living (LIVE/DEAD dye negative) MEC-1 cells after 4 hours of LDC526 incubation. Overlay plots (DMSO and LDC626 1 μM) with adjunct histograms are displayed. A representative plot is shown. (G) Representative histograms of intracellular MCL-1 and BCL-2 staining (with corresponding isotype controls) of MEC-1 cells after 4 hours of LDC626 incubation. (H) Quantification of intracellular MCL-1 and BCL-2 expression (MFI ratio: MFI anti-BCL-2 or anti-MCL-1/MFI corresponding isotype control antibody) after 4 hours of LDC626 incubation (n=3 independent replicates). (I) Increasing LDC526 concentrations decreased the proportion of viable (negativity for Annexin V and DAPI staining, determined by flow cytometry) and increased the proportions of apoptotic (Annexin V+, DAPI-) and dead (Annexin V+, DAPI+) MEC-1 cells (n=4 independent wells; 48 hours incubation). (J) LDC526 dose-response-curves depicting MEC-1 absolute viable cell numbers (n=4 independent wells; 48 hours incubation). (K) Representative plots of MEC-1 cell cycle analysis (flow cytometric BrdU cell cycle assay) after 24 hours of LDC526 incubation. (L) Quantification of MEC-1 cell cycle distribution after 24 hours of LDC526 incubation (n=3 independent replicates). ^* p<0.05; ^** p<0.01; ^*** p<0.001.