Figure 2From: Effect of inhibition of the Ubiquitin-Proteasome System and Hsp90 on growth and survival of Rhabdomyosarcoma cells in vitro 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.Back to article page