Total recovery of Der p 1 after allergen challenge in 20 nonatopic subjects.

<p>*Each subject had two random sampling times</p><p>**Samples taken on one occasion only.</p><p>***Total allergen recovery—comparison between sampling times (p = 0.2; analysis of variance, Friedman test for multiple samples)</p><p>**** Total allergen recovery—comparison between aqueous or particulate at any time point (p≥0.2; Wilcoxon rank sum test)</p><p>N = Number of subjects; IQR = Interquartile Range</p><p>Total recovery of Der p 1 after allergen challenge in 20 nonatopic subjects.</p

Immunohistochemical staining of Der p 1 using mouse anti-Der p 1.

<p>Immunohistochemical staining of Der p 1 using mouse anti-Der p 1.</p

Immunohistochemical detection of Der p 1, IgE and CD68, and the presence of eosinophil leukocytes in nasal biopsies before and after <i>in vivo</i> challenge with Der p 1 allergen.

<p>H-E staining for Eosinophils; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127477#sec006" target="_blank">Methods</a>.</p><p>*Double staining with Der p 1.</p><p>** Staining of cells and the mucous in the epithelium, lamina propria and the submucosal glands. An ordinal scale was used for intensity of Der p 1 staining. This allows for rank order (negative, 1+, 2+, 3+, 4+), but does not allow for measuring relative degree of difference between them.</p><p>***Comparison between non-challenged (pre) and challenged (post) tissue.</p><p>****Comparison between atopic and nonatopic (non-challenged tissue).</p><p>EP: nasal mucosal epithelium; LP: lamina propria; GL: subepithelial mucous glands.</p><p>Number of cells: Mean (range); NA: not available; Statistics: t-test was used for comparison between means.</p><p>Immunohistochemical detection of Der p 1, IgE and CD68, and the presence of eosinophil leukocytes in nasal biopsies before and after <i>in vivo</i> challenge with Der p 1 allergen.</p

Hairpin sequences.

<p>The sequences for the polypurine reverse Hoogsteen hairpins used for specific inhibition of GSTM1, GSTM4 or GSTA4 as well as the scrambled negative control are given.</p

Effect of TMPD or ascorbate (ASC) in combination with MTX on cell viability.

<p>Cells (6×10⁴) were incubated in 1 ml of medium with TMPD (A) or ASC (B) either alone or in combination with MTX at the indicated concentrations, for 3 or 6 days, respectively, and cell viability was determined using the MTT assay. TMPD or ASC were added to the cells 6 h before MTX. Results are expressed as the percentage of surviving cells compared to the control (untreated cells) and represent the mean ± SE of 3 experiments. * <i>p</i><0.05.</p

Endogenous GSH levels and cytochrome c redox capacity in cytosolic extracts.

<p>A) GSH endogenous levels were determined as described in Methods using cytoplasmic extracts from sensitive and MTX-resistant MCF7 cells (6×10⁴). GSH content was calculated in nmols of GSH/mg of total protein (mean ± SE). Results are expressed as the ratio between resistant and sensitive cells. B) Cytoplasmic extracts from sensitive and resistant cells were incubated for 15 min with exogenous cytochrome c (10 µM). A sample with DTT and no cell extract was considered as the maximum value for cytochrome c reduction. The reduction level of cytochrome c was calculated as the difference between the absorbance at 550 nm and at 535 nm. The results are expressed as the percentage of reduction observed in the resistant extracts compared to the sensitive cells and represent the mean ± SE of at least three experiments. * <i>p</i><0.05.</p

Differentially expressed GSTs in MCF7 MTX-resistant cells.

<p>Microarray data analyses were performed with GeneSpring GX 12.0 software as described. For each GSTM isoform, it is expressed the mean of the raw value in sensitive and resistant cells, the fold change in expression after normalization of the data, as well as the corrected <i>p</i>-value after Benjamini-Hochberg FDR filtering.</p

Effect of GSH on the cytotoxicity caused by MTX.

<p>MCF7 sensitive cells (6×10⁴) were plated in 1 ml medium and treated with 1 µM GSH (light grey bars) for 2 h before incubation with MTX. Survival was assessed by the MTT assay 4 days later. Results are expressed as the percentage of survival compared to non-treated cells and represent the mean ± SE of at least 3 experiments. **<i>p</i><0.01, ***<i>p</i><0.001.</p

Effect of veratridine (VERA) and GSH on MTX induced apoptosis.

<p>Cells were incubated with veratridine alone or in combination either with MTX or MTX plus GSH, at the indicated concentrations. Veratridine was added 6 h before MTX. Incubation with exogenous GSH started 8 h before the addition of MTX. In the triple combination, cells were incubated 2 h with GSH, then veratridine was added and 6 h later, treatment with MTX was performed. Apoptosis was assessed 18 h after MTX addition by changes in mitochondrial membrane potential as determined by the Rhodamine 123/Propidium Iodide assay and it is expressed in fold change compared to untreated cells. Results represent the mean ± SE of 3 different experiments. Staurosporine (STP) was used as a positive control. * <i>p</i><0.05.</p

Primer sequences.

<p>The sequences for the forward and reverse primer for detection of GSTM1, GSTM4 and HPRT mRNA levels used for RT-Real Time PCR are given.</p