Selectivity for sensing MGMT activity.

<p>When chemosensor NR-<b>1</b> was added to TK6+ cell lysates (high amounts of MGMT) an immediate increase in fluorescence was observed. Addition of probe to TK6- cell lysates (MGMT knockout) or pretreating TK6+ with 2.5 μM inhibitor PaTrin-2 (10 min, 37°C) led to no change in fluorescence with time. Final probe concentration was 50 nM and total protein used was 200 μg. Data was acquired at 37°C. Measurements were repeated 3 times. Standard deviations are provided in Figure G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.s001" target="_blank">S1 File</a>.</p

Evaluation of MGMT inhibitors with chemosensor 1.

<p>Incubation of purified MGMT enzyme with the inhibitors BG and PaTrin-2 led to a concentration dependent decrease in observed final fluorescence intensity, indicative of MGMT inhibition. MGMT (10 nM) was incubated with inhibitor for 10 min at 37°C in 70 mM HEPES buffer (pH 7.8) containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. Final fluorescence was acquired 10 min after addition of probe (10 nM). Data were normalized to measurements without inhibitor. Each data point is the average of 3 measurements.</p

Performance of modified DNA chemosensors containing the Dabcyl-BG nucleoside.

<p>Fluorescence spectra showing the overall fold-changes in intensity observed when comparing fluorescence measured before (dashed line) and after (solid line) addition of purified MGMT protein are shown at left of each figure. Time courses (on the right) show time-dependent fluorescence increases immediately after addition of enzyme. Final probe and MGMT concentrations were 100 nM. Assays were run at 37°C in 70 mM HEPES buffer pH 7.8 containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. (A) chemosensor <b>1</b> containing dT^FAM, (B), chemosensor <b>2</b> containing Cy3, (C), chemosensor <b>3</b> containing dT^TMR and (D), chemosensor <b>4</b> containing perylene nucleoside. Measurements were repeated 3 times. Standard deviations are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.t001" target="_blank">Table 1</a>.</p

Synthesis of Dabcyl-BG nucleoside phosphoramidite 10, which was used as a monomer in preparation of chemosensors 1, 2, 3 and 4.

<p>Synthesis of Dabcyl-BG nucleoside phosphoramidite 10, which was used as a monomer in preparation of chemosensors 1, 2, 3 and 4.</p

Differential detection of MGMT activity in tumor cell lysates.

<p>Chemosensor NR-<b>1</b> was added to whole cell lysates generated from MCF-7, HT-29 and SW48 cell lines. Final spectra were acquired after a plateau in fluorescence was observed. Spectra were then subtracted from background probe fluorescence (probe alone spectrum) and normalized by total amount of protein in order to compare activity levels. Final probe concentration was 50 nM. Data were acquired at 37°C. Measurements were repeated 3 times. Standard deviations are provided in Figure G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.s001" target="_blank">S1 File</a>.</p

Sequences and optical properties of chemosensors.

<p>Sequences and optical properties of chemosensors.</p

Design principle for a DNA-based chemosensor that reports on MGMT activity.

<p>A short modified DNA oligomer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.t001" target="_blank">Table 1</a>) contains an <i>O</i>⁶-benzylguanine nucleoside modified with a quencher dye. Fluorescence from a neighboring fluorophore (X) is initially quenched as a result of the proximity. When MGMT repairs the alkylated base, the benzyl-quencher group is transferred to the enzyme’s active site and fluorescence emission increases.</p

Parp inhibition rescues CGN sensitivity to MMS.

<p><i>Aag</i>^-/-, WT, and <i>mAagTg</i> neurons are rescued from MMS toxicity (1 mM) after pretreatment with Parp inhibitor Veliparib (A) or Olaparib (B). Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test compared to neurons of the same genotype treated with 1 mM MMS (control). (C) Parp inhibitor Veliparib rescues cell sensitivity at all doses of MMS in WT and <i>mAagTg</i> neurons. Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test comparing sensitivity with or without Veliparib for a genotype at a particular dose of MMS. (D) Expression of BER genes in CGNs. Errors bars denote standard deviation from the mean, <i>Aag</i>^-/- n = 4, WT n = 5, <i>mAagTg</i> n = 1. (E) Aag expression was assessed either before, immediately after, or an hour after MMS treatment using <i>Aag</i> mRNA FISH in WT CGNs. Errors bars denote standard deviation from the mean.</p

Isolation and characterization of <i>Aag</i>^-/- cerebellar granule neurons.

<p>(A) Neurons were isolated from mouse cerebella at post-natal day 5–8. Representative immunocytochemical image depicts neurons (Map2, green) and astrocytes (GFAP, red). Nuclei are shown in blue. (B) Map2+ neurons were quantified from images from 10 pups isolated on different days. Error bars represent standard deviation from the mean. (C) <i>Aag</i> transcripts were counted per cell and are plotted by genotype, where each data point represents one cell. Errors bars denote standard deviation from the mean, n ≥ 50 cells, *** p<0.001 using Student’s standard two-tailed T-test. (D) Cartoon representation of the host-cell reactivation method to measure Aag glycosylase activity in cells. (E) Glycosylase activity over time by cellular fluorescent output from host cell reactivation assay. Solid lines denote mean while dashed lines indicate standard deviation from the mean. (F) WT, <i>Aag</i>^-/- and <i>mAagTg</i> neurons have significantly different Aag glycosylase activity at 24 hours post-transfection. Errors bars denote standard deviation from the mean. WT n = 3, <i>mAagTg</i> n = 2, <i>Aag</i>^-/- n = 2. ** p<0.01 using Student’s standard two-tailed T-test). (G) Fold changes between WT, <i>Aag</i>^-/- and <i>mAagTg</i> Aag expression and glycosylase activity.</p

The majority of the selected proteins modulate the recovery after damage from the three compounds MMS, 4-NQO and t-BuOOH.

<p>A) RNA levels of shRNA targeted genes in 293T cells were measured by qRT-PCR and compared to cells infected with non-silencing control shRNA. B) Survival of cells depleted of target proteins exposed to three DNA damaging agents as revealed by heatmap. The color represents sensitivity to the damaging agent compared to the cell lines with non-silenced targets. ++ indicate high resistance. + low resistance, − high sensitivity, − low sensitivity. C) Knock-down of human homologs of non-toxicity modulating proteins in yeast, as measured by qRT-PCR. D) Survival of cells depleted of human homologs of non-toxicity modulating proteins in yeast. Colors and symbols are the same as in B.</p