Figure 6 (IMAGE)
Caption
Identification of potential regulators of the selective alt-NHEJ inhibition of mibefradil. (A) A schematic representation of the Relative Assessment of DNA Repair (RADAR) plot showing the significance of the different quadrants and their correlation with DNA repair activity. (B) An siRNA screen of 240 siRNAs against common DDR proteins in the EJ-DR assay showing the top hits phenocopying mibefradil in red, the controls siPRKDC (DNA-dependent protein kinase catalytic subunit) and 2 μM NU 4771 (DNA-PK inhibitor) in blue, and 3 μM and 6 μM mibefradil in gray. (C) A focused screen of the deconvoluted siRNAs for the top eleven hits identified in the previous screen in the EJ-DR assay. The top hit simTOR is indicated in red, the controls siPRKDC (DNA-dependent protein kinase catalytic subunit) and 2 μM NU 4771 (DNA-PK inhibitor) in blue, and 3 μM and 6 μM mibefradil in gray. (D) Deconvoluted siRNAs to mTOR reduced mRNA expression of mTOR to ~20% of the non-targeting (NT) control. Gene expression is normalized to a GAPDH control. (n = 3, ***p < 0.001 by Student's T test) (E) Dose-response curve of mTORC1/2 inhibitor (mTORC1/2i) in the EJ-DR assay showing alt-NHEJ activity in red, HR activity in green, and cytotoxicity (CT) in blue. mTORC1/2i showed a selectivity index (SI), calculated as CT IC50/alt-NHEJ IC50, of 7.22. (F) Dose-response curve of Akt1/2-inhibitor (Akt-1/2i) in the EJ-DR assay. Akt-1/2i showed an SI of 77.67. Data are represented as mean ± SEM.
Credit
Correspondence to - Yulia V. Surovtseva - yulia.surovtseva@yale.edu and Ranjit S. Bindra - ranjit.bindra@yale.edu
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Copyright: © 2021 Paradkar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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