Figure 3.

Roniciclib induces growth arrest and cell differentiation in neuroblastoma cells. (A) IMR-32, ACN and SH-SY5Y neuroblastoma cell lines were cultured in presence of various concentrations of Roniciclib for 24, 48 and 72 h. At each harvest point, cells were trypsinized and counted in Trypan blue. Untreated cells (Roniciclib 0 µM) were cultured with 0.1% DMSO. Arrows indicate the Roniciclib concentrations chosen for each cell line after 72 h, which gave significant effects without toxicity. Data are representative of three independent experiments ± SD (*** p < 0.001). (B) Proliferation rate evaluated by immunofluorescence analysis of IMR-32, ACN and SH-SY5Y neuroblastoma cell lines untreated (Roniciclib 0 µM) or treated with Roniciclib 1 µM, 20 µM and 5 µM respectively for 72 h, using the anti-Ki-67 (red) antibody. Cells were counterstained with DAPI to visualize nuclei (blue). (Scale bar: 20 µm). (C) Morphological characteristics of IMR-32, ACN and SH-SY5Y cell lines untreated (Roniciclib 0 µM) or treated with Roniciclib 1 µM, 20 µM and 5 µM respectively for 72 h. Roniciclib treated cells displayed a reduced growth rate, increase in cell size, elongated shape and emission of neurite-like extensions (arrows) (Scale bar: 40 µm). In the insets are visualized enlargements of particular examples of neurite-like protrusions produced by each cell line. Histograms represent the percentages of cells extending neurite-like protrusions among untreated (U) or Roniciclib treated (R) cells after 72 h. Data are representative of three independent experiments ± SD (*** p < 0.001).