Fig. 5From: Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibitionEffects of crizotinib treatment on autophagy, migration, invasion and colony formation in RH4 and RH30 cell lines. a Acridine orange staining showed that RH4 and RH30 DMSO-treated control cells exhibited no autophagosomes, whereas crizotinib-treated cells showed abundant cytoplasmic acidic vesicular organelles (AVO) formation, a characteristic of autophagy. Samples were observed under an ApoTome fluorescence microscope. b LC3 and p62 protein levels were examined in RH4 and RH30 cells treated with crizotinib (Crz) at various concentrations for 24 h. Control cells (Ctrl) received DMSO vehicle. c ARMS cells were treated with crizotinib (1.5 and 5 μM) for 24 h and H2DCFDA was added 45 min before collecting cells. H2DCFDA fluorescent intensities were analysed by FACSCalibur. Control cells (Ctrl) with H2DCDA staining were used as a negative control. Bar charts represent the mean fluorescence ± SD for each sample (*, p-value <0.05 compared with the respective negative control; **, p < 0.01). Three independent experiments were performed. d Crizotinib (1.5 μM) treatment led to a decreased cell migration in both RH4 and RH30 cells. Representative images of migrated cells using the transwell migration assay (crystal violet staining, magnification of 20×). Histograms represent the average values ± SD as determined by counting 8 fields of cells under the microscope. Three independent experiments were performed, each in triplicate (*, p-value <0.05 compared with the respective negative control; **, p < 0.01). Control cells were treated with DMSO. e Crizotinib (1.5 μM) treatment led to decreased cell invasion in both RH4 and RH30 cells. Control cells were treated with DMSO alone. Representative images of invasive cells using the Matrigel-invasion assay (crystal violet staining, 20× magnification). Histograms represent the mean value ± SD as determined by counting 8 fields of cells under the microscope. Three independent experiments were performed, each in triplicate (*, p < 0.05). f Crizotinib (1.5 μM) induction led to a decreased cell colony formation capacity in both RH4 and RH30 cells. Control cells were treated with DMSO alone. Representative images of a single cell derived-sphere in mocked control and treated cells cultured in soft agar for 21 days (original 40× magnification)Back to article page