Figure 5.
Inhibition of GR Decreases Growth and Survival of Ewing Sarcoma Cells
(A) Cell viability assays applied the MTT method on three Ewing sarcoma lines (CHLA9, A673, and RD-ES), which were treated for 24 h with increasing concentrations of RU486.
(B) Increasing concentrations of DO6, a non-steroidal GR antagonist, were incubated with A673 cells as in (A), and an MTT assay was performed in triplicates. ∗p < 0.05.
(C) A673 cells were transfected with either FLI1-specific or control-scrambled siRNAs (si-C). EWS-FLI1 knockdown efficiency was tested after 48 h using immunoblotting.
(D) FLI1-silenced A673 cells (8 × 103) were seeded in 96-well plates and treated with either vehicle or RU486 (10 μM). MTT assays were performed after 48 h in quadruplets. ∗∗p < 0.01; ∗∗∗p < 0.001.
(E) A673 cells were seeded in 100-mm dishes. Thereafter, cells were treated for 48 h with DEX (1 μM), RU486 (10 or 20 μM), or the combination. Shown are results of an apoptosis assay performed using an annexin V/7-AAD kit (BioLegend). Quantification of the fractions of early and late apoptotic cells is shown. The experiment was repeated twice.
(F) The indicated cells were sparsely seeded in 6-well plates. Cells were later treated every other day with either vehicle, DEX (1 μM), RU486 (10 μM), or the combination. Ten days later, cells were fixed and stained with crystal violet. Photos are shown along with bar plots presenting the quantification of colonies in 5 non-overlapping microscope fields. The experiment was repeated twice. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.