Effect of combined treatment with CX compounds and chemotherapeutic drugs.

<p>S-CEM and R-CEM viability was assessed by the MTT method after 24 h treatment with increasing concentrations of both CX-4945 and Vbl, administrated alone or in combination, at fixed ratio (1 µM CX-4945 : 0.8 µg/ml Vbl for S-CEM, 1 µM CX-4945 : 2 µg/ml Vbl for R-CEM, due to their different sensitivity to Vbl). Viability (mean values ± SE of four experiments) was plotted as function of Vbl concentrations (left panels), or CX-4945 concentrations (right panels).</p

Apoptosis induction by CX-4945 and CX-5011.

<p>(A) Apoptosis was assessed by evaluation of nucleosomes present in the cytosol, using the Cell Detection Elisa kit (Roche), after 8 h of CEM cell treatment as indicated. Nucleosome enrichment was calculated from the ratio between the signal in treated and untreated cells; reported values are the means ± SE of four independent experiments. (B) Caspase-dependent PARP cleavage was analyzed by WB on 10 µg proteins of lysate from CEM or U2OS cells treated as indicated. Actin WB was used as loading control. Representative WB of three independent experiments are shown. f.l. PARP: full length PARP.</p

CK2 activity in CX-4945-treated cells.

<p>(A) CK2 activity was measured towards a synthetic specific peptide. 1–2 µg of proteins from total lysates of the indicated cells were incubated with the peptide and a radioactive phosphorylation mixture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#s2" target="_blank">Materials and Methods</a>). Activity is reported as percentage of that found in vehicle-treated control cells. Mean ± SE values of four independent experiments are shown. (B) 10 µg of total proteins were analyzed with the indicated antibodies; actin was used to normalize the loading. Representative WB of three independent experiments are shown.</p

Pgp expression.

<p>10 µg of proteins from total lysates of the indicated cells were loaded onto an SDS-PAGE, blotted and analyzed by WB with anti-Pgp or anti-actin.</p

Cell viability of CX-4945 and CX-5011 treated Imatinib-resistant cells.

<p>See legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g005" target="_blank">Figure 5</a> for details.</p

Protein phosphorylation in lysates from cells treated with CX-4945, CX-5011, or staurosporine.

<p>S-CEM (upper panel) or R-CEM (lower panel) were treated with the indicated concentrations of the CX inhibitors or staurosporine for 16 h, then lysed. 5 µg of total proteins were incubated with a radioactive phosphorylation mixture, resolved by SDS-PAGE, blotted, and analyzed by digital autoradiography (radioactivity). WB for actin was used as loading control. Asterisk * denotes samples where the inhibitors were added in vitro during the phosphorylation assay (not administrated to the cells). Representative results of five independent experiments are shown.</p

DC₅₀ of CX-4945 and CX-5011.

<p>The values, calculated from 48 h MTT assays reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g005" target="_blank">Figures 5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g008" target="_blank">8</a> (except for CEM* = 24 h treatment), are the mean of 4–6 independent experiments ± Standard Deviation. FCS concentration (v/v) during the treatment is also indicated.</p

CK2 activity in CX-5011-treated cells.

<p>S-CEM and R-CEM cells were treated for 24 h with different concentrations of CX-5011. Cell lysates were analyzed for the activity of CK2 towards a synthetic specific peptide (upper graph), or by WB with the indicated antibodies (lower panel, separate development for S- and R-CEM cells). See details in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g002" target="_blank">Figure 2</a>.</p

Effect of CX-4945 on doxorubicin accumulation.

<p>CEM cells were preincubated for 30 min with vehicle (Contr) or CX-4945 at the indicated concentrations, then further incubated for 30 min with 25 µM doxorubicin, whose amount, detected fluorimetrically as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#s2" target="_blank">Materials and Methods</a>, is reported as percentage of the value found in control S-CEM (mean ± SE of three independent experiments).</p

Cell viability of CX-4945 and CX-5011 treated 2008 cells.

<p>See legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049193#pone-0049193-g005" target="_blank">Figure 5</a> for details.</p