Induction of autophagy in ARMS and ERMS cell lines. (A) To assess apoptosis and autophagy induction, PARP and LC3 protein expression were determined by Western blot analysis, after 48 h-treatment with Bortezomib (7,5nM), 17-DMAG (50nM), or the combination Bortezomib/17-DMAG, using β-actin (actin) as loading control. (B) Under these conditions, analysis of RH30 and RD cell morphology and chromatin integrity was performed, using a contrast-phase microscope (63x magnification) and processing cells for DAPI/γ-tubulin staining. (C) Effects of single-agent or combinatorial treatments on vacuoles acidification were investigated by staining RH30 and RD cells with acridine orange (5 μg/mL), in the presence or absence of lysosomal inhibitor chloroquine. (D) Western blot analysis of cleaved PARP and processed LC3 (LC3-II) proteins in RH30 and RD cells treated with 48 hours with Bortezomib (7.5nM), 17- DMAG (50nM), or their combination, in the presence or absence of cloroquine or rapamycin. Proteins were analyzed by SDS-PAGE and band densities were measured with NIJ image software. Values are expressed as folds of control and are means ± standard deviation of three independent experiments.