Thermogram feature parameters of each clinical group, including “healthy” and non-MGUS sera controls.

<p>^a Median value (lower and upper value in the first (25th) and third (75th) quartiles).</p><p>T_max, temperature of the peak maximum.</p><p>Cp1^ex, excess specific heat capacity of the first thermal transition.</p><p>Cp2^ex, excess specific heat capacity of the second thermal transition.</p><p>Cp1^ex/Cp2^ex, ratio of the excess specific heat capacities of the first and second transitions.</p><p>T_FM, first moment temperature.</p><p>Thermogram feature parameters of each clinical group, including “healthy” and non-MGUS sera controls.</p

Box chart representations of thermogram feature parameters.

<p>For each panel, boxes from left to right represent serum samples from (a) healthy controls, (b) MGUS-κ, (c) MGUS-λ, (d) All-MGUS (which compiles MGUS-κ and MGUS-λ samples together) and (e) non-MGUS. The bottom and top box edges indicate the 25^th and 75^th percentiles. Within the box, the median value is indicated by the horizontal line, and the mean by the cross inside the box. The whiskers extend from the ends of the boxes to the minimum and maximum experimental values.</p

Patient demographics and disease characteristics.

<p>^a All serum samples were from white people.</p><p>^bMGUS encompasses serum samples of the following isotypes: IgG κ (10 samples), IgG1-κ subclass (2), IgA κ (2), IgM κ (2) IgG λ (7), IgA λ (3), IgM λ (2).</p><p>^cNon-MGUS includes serum samples from patients with subjacent immunological pathologies who were ruled out of having MGUS through serum immunofixation.</p><p>Patient demographics and disease characteristics.</p

DSC thermograms of serum samples from healthy individuals and patients with MGUS-κ.

<p>Samples were gathered as MGUS-κ based on the κ light chain that was found associated with different monoclonal immunoglobulin heavy chains. (A) A set of thermograms obtained from six healthy (control) serum samples. The panel shows an average healthy control plot (dashed brown line) and the standard deviation (shadow). (B) A set of thermograms of serum samples obtained from MGUS patients with IgG κ subtype. (C) Another set of themograms obtained from individuals with IgG κ subtype. For the ease of visualization, thermograms were clustered in either panel B or C based on the similarity of their DSC profiles. (D) Thermograms from patients having IgG1-κ subclass. (E) Thermograms from patients having IgA κ (black) or IgM κ (red) subtypes. For the sake of comparison, the average healthy (control) thermogram (dashed brown line) is presented in all the panels.</p

DSC thermograms of serum samples from patients with MGUS-λ.

<p>Samples were gathered as MGUS-λ based on the λ light chain that was found associated with the different monoclonal immunoglobulin heavy chains. (A) A set of thermograms of serum samples obtained from patients with IgG λ subtype. (B) A set of thermograms from individuals with MGUS IgG λ subtype (black) or IgM λ subtype (red) grouped together to highlight their similar shape. (C) Several thermograms from patients having IgA λ (continuous black and red plots, and dashed black plot) or IgM λ (dashed red plot) subtypes grouped together because they show similar shapes. For the sake of comparison, the average healthy (control) thermogram is presented in all the panels.</p

Combination of nilotinib, but not imatinib, with doxorubicin displayed a synergistic effect on apoptosis in leiomyosarcoma SK-UT-1 cells.

<p>A, <i>left</i>, DNA content of cells was measured by flow cytometry. Representative histograms of vehicle-treated (DMSO), DXR (0.05 µM)-treated, nilotinib (5 µM)-treated and pre-nilotinib 24 h+DXR-treated cells are shown. The fluorescence values used to calculate the peak corresponding to the sub-G1 phase are indicated on each histogram. <i>Right</i>, Columns show percentage of apoptotic cells in the absence (vehicle, V) or presence of DXR and nilotinib as single agents or combined (pre-Nilotinib+DXR). Each column represents mean ± SEM of four independent experiments. ^**<i>P</i><0.01 and ^***<i>P</i><0.001 versus DXR-treated cells. B) Antiproliferative effect of imatinib as single compound or combined with DXR (0.05 µM) (pre-Imatinib+DXR) for 72 h. Each column represents mean ± SEM of three independent experiments. C) Panels show the immunoreactive bands of procaspase 8 and 3, cleaved caspase 8 and 3, and PARP fragmentation in representative immunoblots of leiomyosarcoma cells treated as in A.</p

Differences in thermogram feature parameters across the serum sample groups.

<p>The thermogram feature parameters are identified at the top of each panel. The analysis of the data by a non-parametric Kruskal-Wallis test revealed significant differences in the six thermogram parameters displayed in the figure (Area, p = 9.82E-3; T_max, p = 8.73E-3; Cp1^ex, p = 5.59E-3; Cp2^ex, p = 4.26E-4; Cp1^ex/ Cp2^ex, p = 5.92E-4; T_FM, p = 2.16E-3), indicating they can be used to distinguish the different serum groups, and also validated the utilization of post-hoc pairwise comparisons. Differences between the serum sample groups were examined for every thermogram parameter by the unpaired Mann-Whitney <i>U</i> test. The figure panels indicate “yes” or “no” to specify whether statistically significant differences occurred (the actual p values are shown in the panels).</p

Nilotinib, but not imatinib, increased intracellular doxorubicin in human STS cells.

<p>Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were incubated with vehicle (DMSO) or DXR (1 µM) in the absence (vehicle, V) or presence of nilotinib (1–10 µM) or imatinib (1–10 µM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037735#s2" target="_blank">Material and Methods</a> section. After 24 h incubation, intracellular DXR was measured by its fluorescence intensity with flow cytometry. <i>Left</i>, Median of intracellular DXR fluorescence in the absence (V) or presence of TK inhibitors normalized to vehicle-treated cells (taken as 100%). Each column represents mean ± SEM of seven independent experiments. <i>Right</i>, Representative flow cytometry analysis of the intracellular DXR fluorescence detected with excitation at 488 nm and emission at 580 nm in both cell lines. ^***<i>P</i><0.001 versus DXR-treated cells.</p

Nilotinib decreased fetal bovine serum-induced AKT activation and fully blocked ERK1/2 and p38 MAPK activation.

<p>Sub-confluent synovial sarcoma SW982 cells were deprived of fetal bovine serum (FBS) for 4 h. Cells were then stimulated for 30 min with 10% FBS in the absence (vehicle, V) or presence of imatinib (10 µM) or nilotinib (5 and 10 µM) as single compounds. <i>Upper panels</i> show representative immunoblots of four independent experiments. Columns represent the phosphorylated ratio of AKT, ERK1/2 and p38 MAPK. Each column represents mean ± SEM of four independent experiments normalized to FBS-depleted cells (-FBS, taken as 100%). ^*<i>P</i><0.05; ^**<i>P</i><0.01 and ^***<i>P</i><0.001 versus vehicle-treated cells stimulated with FBS.</p

Combination of nilotinib, but not imatinib, with doxorubicin (DXR) displayed a synergistic effect on growth inhibition and apoptosis in synovial sarcoma SW982 cells.

<p>A) Antiproliferative effect of nilotinib as single compound or combined with DXR. Synovial sarcoma cells were treated with vehicle or nilotinib (0.1–40 µM) alone or combined simultaneously with DXR (0.1, 0.3 and 0.5 µM) for 72 h. Each value represents mean ± SEM of four individual experiments performed in triplicate. B) Subsequent isobolographic analyses were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037735#s2" target="_blank">Material and Methods</a> section. Drug combination was synergistic at all concentrations (CI<1), as shown in the isobologram. C) Apoptotic effect of nilotinib as single compound or combined with DXR (0.1 µM) for 72 h. <i>Upper panels</i> show the immunoreactive bands of procaspase 3, cleaved caspase 3 and PARP fragmentation in representative immunoblots. DNA content of cells was measured by flow cytometry. Columns show percentage of apoptotic cells in the absence (vehicle, V) or presence of DXR and nilotinib as single compounds or combined (DXR+nilotinib). Each column represents mean ± SEM of four independent experiments. ^***<i>P</i><0.001 versus DXR-treated cells. D) Antiproliferative effect of imatinib (0.5–10 µM) as single compound or combined with DXR (0.1 µM) for 72 h. Each column represents mean ± SEM of three independent experiments.</p