MEK inhibition reduces viability of MPNST cells.

<p>Viability of MPNST cells under MEKi treatment was monitored by MTT. MPNST cells were treated with different doses of MEKi U0126 (upper panel) or PD0325901 (lower panel). T265 cells and S462 cells highly responded to MEKi treatment with reduced viability, whereas NSF1 cells did not show enhanced sensitivity to MEKi treatment, compared to untreated control cells (black bars). Maximum reduction of viability was achieved at 10 μM of U0126 and 1 μM of PD0325901 (reduction down to 53% ± 7% and 2% ± 1% in T265 cells, and 59% ± 7% and 19% ± 11% in S462 cells) (mean value of 3–5 measurements in each cell line (A). Relative ERK-P expression in T265 cells after MEKi treatment was analyzed. Western blots of ERK-P, ERK and GAPDH antibodies are demonstrated exemplarily for T265 cells after treatment with 10 nM PD0325901 (+) compared to untreated cells (-). T265 cells treated with MEKi demonstrated strongly reduced phosphorylated ERK level (grey bar, 0.32-fold ± 0.11-fold) compared to untreated control cells (black bar, 1.00-fold ± 0.25-fold). PD0325901 treatment did not affect total ERK level in treated (grey bar, 1.04-fold ± 0.07-fold) versus untreated T265 cells (black bar, 1.00-fold ± 0.11-fold) (mean value of 3 repeated measurements).</p

ATRA treatment reduces migration in 2/3 MPNST cell lines.

<p>Migration of MPNST cells under ATRA treatment, monitored using the xCELLigence system. Area under curve (AUC) was calculated after 16 h. Bars indicate AUC values (mean of 3–5 repeated measurements within each cell line) compared to control cells. T265 cells (25 μM ATRA) showed strongly reduced migration down to 37% ± 15%. A minor effect of ATRA treatment was observed in S462 cells (10 μM ATRA) with reduced migration of 79% ± 7%. No effect on migration was observed in NSF1 cells (50 μM ATRA) (105% ± 5%) (A). Migration curves of ATRA treated T265 cells (ATRA, grey line) and untreated T265 cells (control, black line) are demonstrated exemplarily (B).</p

Therapeutic pattern and gene expression profiles in MPNST cells.

<p>Relative expression, functional effects and promoter methylation is depicted in a semi-quantitative manner applying the following score: three symbols—highest, two symbols—medium, one symbol—low, dash—no, induction/reduction/expression among the three MPNST cell lines.</p

ATRA treatment induces apoptosis in T265 and S462 cells.

<p>Flow cytometry analysis (FACS) of TUNEL staining in ATRA treated MPNST cells at day 7 (5 μM). Bars represent intensity values (mean of 2–3 repeated measurements within each cell line) compared to control cells (black bars): Increase of TUNEL staining intensity to 127% ± 10% in T265 cells, to 374% ± 35% in S462 cells and 109% ± 3% in NSF1 cells (A). Right shift (regular line) in fluorescence intensity compared to untreated controls (dashed line) in the FACS histogram is shown for T265, S462 and NSF1 cells (B).</p

Combined ATRA and MEKi treatment adds reduced viability.

<p>Relative viability of MPNST cells after combination therapy (day 5) was assessed by MTT assay. MPNST cells were treated with ATRA (light grey) and MEKi (dark grey) alone or in combination (striped). PD0325901 combination treatment is shown in the upper panel, U0126 combination treatment in the lower panel. A significant reduction of viability in the combination therapy group was found for T265 cells and S462 cells with both inhibitors, compared to mono-treated cells. Bars represent mean values of repeated (3–5 times) measurements in each cell line). NSF1 cells displayed no additive effects due to combination-therapy, neither with PD0325901 nor with U0126, as well as a very weak response to monotherapy.</p

ATRA therapy affects viability of MPNST cells in a dose-dependent manner.

<p>Viability of cells under ATRA treatment was monitored by MTT assay on day 7 of treatment. T265 cells showed reduced viability at high ATRA concentrations (25 μM and 50 μM) and enhanced viability at low ATRA concentrations (5 μM and 10 μM). S462 cells demonstrated reduced viability at all ATRA concentrations. MCF-7 cells used as control were sensitive to ATRA treatment at all concentrations. In contrast, normal rat Schwan cells displayed no change of viability under ATRA treatment. Bars represent mean values of 3–4 repeated measurements within each cell line.</p

ATRA treatment induces expression of S100 and inhibits PMP22 expression.

<p>Flow cytometry analysis (FACS) of S100 and PMP22 protein expression in ATRA treated MPNST cells on day 7 of treatment (5μM). Bars indicate increase or decrease of fluorescence intensity (%) compared to untreated controls (100%). Values represent mean values (± SD) of 2–4 repeated measurements within each cell line: T265 cells showed an increase of 158% ± 19%, S462 cells of 71% ± 18% and NSF1 cells of 49% ± 33% (A). T265 cells showed a reduction of PMP22 expression of 22% ± 10%, and S462 cells of 20% ± 14% (B).</p

ATRA treatment induces expression of classical targets in MPNST.

<p>Relative mRNA expression of bona fide ATRA targets was analyzed in MPNST cells after ATRA treatment by qRT-PCR. Bars represent levels of <i>CRABP2</i>, <i>CYP26A1</i> and <i>RARB</i> relative to untreated control cells (black line, 1-fold). All three MPNST cell lines showed induction of the investigated targets. T265 cells showed lowest induction (<i>CRABP2</i>, <i>CYP26A1</i>, <i>RARB</i>) by ATRA treatment (1.5-fold ± 0.1, 55.8-fold ± 21.1, 10.8-fold ± 3.3) whereas NSF1 cells showed highest induction of mRNAs after ATRA treatment (57.8-fold ± 26.2, 633.0-fold ± 43.6, 24.5-fold ± 6.1) among the three MPNST cell lines. Measurements were repeated in each cell line 3 times.</p