Measurement of mitochondrial membrane dysfunction among TW1 and TW17 isolates.

<p>For the flow cytometry analysis, cells were stained with 100 nM MitoTracker Red CMXRos (which fluoresces upon oxidation in respiring mitochondria). (A) Quadrant plots, (B) histograms and (C) bar graph of flow cytometry analysis between TW1 and TW17 are depicted (n = 4). (D) The red fluorescence of MitoTracker Red CMXRos was also visualized by confocal microscopy.</p

FLC exposure alters CW integrity in the sequential isolates of C. albicans.

<p>(A) Sequential isolates were tested with CW perturbing agents like Triton X-100 (upto 0.04%), 0.02% SDS, 16 µgml⁻¹ Congo Red. (B) Susceptibility to Calcofluor White (upto 50 µgml⁻¹) was tested. These spot tests were performed as described in methods. (C) Passive diffusion rate of PI was monitored by spectrofluorimeter. Briefly, the cells were pre-incubated with 3 µM PI for 45 min and then centrifuged. The supernatant was taken and the emission spectrum of PI was recorded for each strain. (D) The cells with PI were subjected to flow cytometry analysis to measure the amount of PI accumulated in each strain. The data is represented as the mean fluorescent intensity (n = 2).</p

Accumulation of odd chain-FA containing PGLs in the FLC resistant isolates.

<p>Total amount of odd chain FA-containing PGL was calculated by adding the normalized amounts of each odd chain FA containing PGL molecular species (namely 31-C, 33-C, 35-C and 37-C containing PGLs). The data is represented as % of total PGL + SL + SE mass spectral signal after normalization to internal standards. Values are mean of 3 independent analyses (n = 3). Asterisks “*” represents p < 0.05. Lipid data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s004" target="_blank">Sheet S1</a> (worksheet 3), <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s006" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s008" target="_blank">Table S3</a>.</p

Upregulation of the sterol biosynthetic pathway in AR isolates.

<p>(A) Changes in the composition of major sterols and its intermediates among the sequential isolates of <i>C. albicans</i> were analyzed as described in methods. Green and black colour of gene in the pathway (A) represents upregulation and no change, respectively. (B) Total SEs are represented as % of the total PGL+ SE + SL mass spectral signal after normalization to internal standards and were determined as described in methods. (C) The fold change in gene expression levels of various <i>ERG</i> genes (relative amplification to <i>ACT1</i>) between TW1 (most susceptible to FLC) and TW17 (most resistant to FLC) were determined by RT-PCR. The gel picture is a representative of the RT-PCR analysis performed in replicates. Values in the histogram are means ± SD (n = 3 for all <i>Candida</i> strains). Asterisks “*” represents p<0.05. Lipid data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812.s004" target="_blank">Sheet S1</a>, worksheet 3 and 4.</p

FLC exposure leads to gradual development of a partially compromised CW and affects PG biosynthesis in several other clinical isolates of <i>C. albicans</i>.

<p>A. Several pairs of isogenic clinical isolates of <i>C. albicans</i> were tested with 0.02% SDS, a CW perturbing agent. The spot tests were performed as described in methods. B. PL analysis of various AS/AR clinically matched isogenic isolates by HP-TLC. Briefly, cells were grown overnight (to the exponential growth phase) in YEPD medium. Lipids were extracted as described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039812#pone.0039812-Bligh1" target="_blank">[18]</a> and separated by thin-layer chromatography using chloroform: methanol: acetic acid (65:28:8) as the solvent system. HP-TLC analysis of each match pair and the corresponding values of each lipid class as mol % are also indicated.</p

Evidence for Abasic Site Sugar Phosphate-Mediated Cytotoxicity in Alkylating Agent Treated <em>Saccharomyces cerevisiae</em>

<div><p>To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in <em>Saccharomyces cerevisiae</em>, we replaced the <em>RAD27^FEN1</em> open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In <em>S. cerevisiae</em>, Rad27^Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in <em>S. cerevisiae</em>, and this partially rescued the MMS hypersensitivity observed with <em>rad27^fen1</em>-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in <em>rad27^fen1</em>-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27^Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.</p> </div

Rescue of CPT and ionizing radiation sensitivity by human Pol β dRP lyase.

<p>(A) Ten-fold dilutions of each indicated strain were spotted onto plates containing either DMSO alone (mock, lanes 1–5) or 25 µM CPT (lanes 6–10). <i>POLβ</i> strains were not sensitive to CPT, an agent that produced 3′ blocking groups. <i>mre11</i>Δ and <i>rad50</i>Δ deleted strains were used as positive controls for CPT-sensitivity. (B) <i>POLβ</i> strains do not display sensitivity to ionizing radiation (IR), an agent that produces predominately 3′ blocking groups. As in (A), <i>mre11</i>Δ and <i>rad50</i>Δ deleted strains were used as positive controls for IR sensitivity. Indicated strains were plated and treated as described in Material and Methods, and were mock-treated (lanes 1–5) or subjected to 200 Gray (Gy) of IR, lanes 6–10.</p