The TDP1-biosensor had the sequence 5��-ATTO488-AAA GCA GGC TTC AAC GCA ACT GTG AAG ATC GCT TGG GTG CGT TGA AGC CTG CTT T-BHQ1-3��.2.3. Cell Culture and Extract PreparationCaco2 cells were grown in minimum essential medium (MEM) supplemented with 20% FBS, 1% non-essential amino acids, 1% PenStrep. HT29 cells were grown in McCoy 5A Ganetespib FDA medium supplemented with 10% FBS, 1% PenStrep. The cell cultures were maintained in a humidified Inhibitors,Modulators,Libraries incubator (5% CO2/95% air atmosphere at 37 ��C). Cells were harvested by trypsin treatment (0.25% Trypsin-EDTA solution). The trypsin was inactivated with FBS-containing media, followed by two consecutive washes with 1�� PBS. Cells were counted, aliquoted into tubes each containing 5 �� 105 cells, and stored at ?80 ��C until further analysis.

Preparation of whole cell extracts was done by mixing 5 �� 105 cells with 500 ��L, 250 ��L, or 100 ��L lysis buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 Inhibitors,Modulators,Libraries mM DTT) for hTopI activity measurement, hTopI activity measurement in the presence of CPT, and TDP1 activity measurement, respectively. Tubes with cells and lysis buffer were incubated on ice for 10 min before the extract was used.2.4. Western Blot Analysis of hTopI in Cell ExtractsCell extracts from Caco2 or HT29 cells were analyzed by 10% SDS-polyacrylamide gel-electrophoresis followed by western blotting onto a Nitrocellulose Protran BA85 (GE Healthcare Life Sciences, Broenby, Denmark) membrane Inhibitors,Modulators,Libraries and antibody incubation using a polyclonal hTopI antibody (Topogen, Port Orange, FL, USA) and a Anti-TATA binding protein TBP [1TBP18] antibody (Abcam, Cambridge, UK) as loading control.

2.5. Detection of hTopI Activity by REEAD AssayThe hTopI reaction was carried out in a 20 ��L reaction volume containing a divalent cation depletion buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT) supplemented with S(hTopI)Id16 DNA substrate to a final concentration of 0.5 ��M. Reactions were Inhibitors,Modulators,Libraries initiated by the addition of 10 ��L cell extract in the presence or absence of CPT as indicated in the figure legends. DMSO was added as solvent control of CPT. Incubation was continued at 37 ��C for 60 min for the dilution experiments and 15 min for CPT sensitivity experiments, before the heat inactivation of the Entinostat enzymes by incubation for 5 min at 95 ��C.

To allow quantification, 5 nM of ligated S(PosC)Id33 was added to the heat inactivated reaction mixture and to increase the efficiency of hybridization NaCl was added to a final concentration of 250 mM. Preparation of the ligated S(PosC)Id33 was done using the T4 DNA ligase, and the resulting ligated circles were exonuclease selleckchem digested with ExoI and ExoIII to remove non-ligated S(PosC)Id33 before gel-purification using 8% polyacrylamide gel. The concentration of the obtained circles was determined by spectrophotometric measurement.