Figure 7.

Roniciclib limits nucleolar proteins expression without influencing neuroblastoma cells apoptosis. (A, C) Protein lysates from IMR-32, ACN and SH-SY5Y cell lines untreated (u = Roniciclib 0 µM) or treated with Roniciclib (R) 1 µM, 20 µM and 5 µM respectively for 72 h were collected and subjected to Western blot analysis with anti-NCL, anti-NPM1, anti-GPC2, anti-PES1, anti-PARP (c = cleaved PARP), and anti-phospho-Akt (P-Akt) antibodies. Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Supplementary Figure S7, panels 1–2, shows the entire blots images. (B, D) Protein levels of the Roniciclib treated cells were quantified by densitometry, normalized to those of the untreated cells (fold induction = 1) and to the content of the loading control protein (Actin or total Akt), then visualized by histograms. Data are representative of three independent experiments ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001). (E) Immunofluorescence analysis of IMR-32, ACN and SH-SY5Y cell lines untreated (Roniciclib 0 µM) or treated with Roniciclib for 72 h, using TUNEL staining (green) to reveal DNA strand breaks generated during apoptosis. NB cells pre-treated with DNase I were used as positive controls. Cells were counterstained with DAPI to visualize nuclei (blue). (Scale bar: 20 µm).