Fig. 6.

Inhibition of FKBPs impairs homologous recombination independently of mTOR. (A) A schematic presenting the strategy for chemical inhibition of FKBP25 by rapamycin without inhibiting mTOR. mTOR activity is restored by competition with FK506, which has a similar affinity for FKBP12 as rapamycin. [The following Protein Databank (PDB) entries were used in the generation of this figure; PDB ID 3FAP (Liang et al. 1999) and PDB ID 2MPH (Prakash et al. 2016)] (B) MTT proliferation assay as a proxy for the restoration of mTOR activity measuring proliferation. Cells were treated with either a dimethyl sulfoxide (DMSO) control, 10 nmol/L rapamycin, or 10 nmol/L Torin1 in the absence or presence of 2 μmol/L FK506. Error bars represent the standard error of 4 measurements across 4 independent experiments. (C) MTT proliferation assay of cells treated with increasing doses of the Parp-inhibitor olaparib in combination with 10 nmol/L rapamycin or 10 nmol/L rapamycin and 2 μmol/L FK506. Error bars represent the standard error of 4 measurements across 4 independent experiments. (D) MTT proliferation assay, cells treated as in (E). Error bars represent the standard error of 4 measurements from 2 independent experiments. (E) Flow cytometry reporter assay measuring the homolous recombinant double strand break repair pathway utilization in cells treated with DMSO control; 10 nmol/L rapamycin, 10 nmol/L Torin 1, and 2 μmol/L FK506; or 10 nmol/L rapamycin and 2 μmol/L FK506. Error bars represent standard error of 4 independent experiments. Significance relative to the DMSO control treated cells is shown above each bar. *, P < 0.05; **, P < 0.01.