roniciclib (bay 1000394)  (Stegmann Systems GmbH)

Journal: Scientific Reports

Article Title: Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

doi: 10.1038/s41598-020-69499-6

Figure Lengend Snippet: Roniciclib interferes with cancer stem cell markers and induces nucleolar fragmentation in neuroblastoma cells. 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 anti-CD44v6, anti-CD114, anti-NCL, anti-NPM1, anti-GPC2 and anti-PES1 (all green) antibodies. White arrows indicate nucleolar fragmentation with nucleoplasmic/cytoplasmic redistribution of NPM1 and PES1 proteins. Cells were counterstained with DAPI to visualize nuclei (blue). (Scale bars: 20 µm).

Article Snippet: Roniciclib (BAY 1000394), by AdooQ Bioscience (Irvine, CA, USA), was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM and maintained at − 20 °C until used.

Techniques: Immunofluorescence

Journal: Scientific Reports

Article Title: Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

doi: 10.1038/s41598-020-69499-6

Figure Lengend Snippet: 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).

Article Snippet: Roniciclib (BAY 1000394), by AdooQ Bioscience (Irvine, CA, USA), was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM and maintained at − 20 °C until used.

Techniques: Cell Differentiation, Cell Culture, Immunofluorescence, Produced

Journal: Scientific Reports

Article Title: Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

doi: 10.1038/s41598-020-69499-6

Figure Lengend Snippet: Roniciclib impairs neuroblastoma neurospheres formation ability. ( A ) Microscopic analysis of neurospheres produced by IMR-32, ACN and SH-SY5Y cells untreated (Roniciclib 0 µM) or treated with Roniciclib 1 µM, 20 µM and 5 µM respectively, for 72 h, and cultured for three days in serum-free medium maintaining the same Roniciclib concentrations. (Scale bars: 200 µm on the left of both panels; 100 µm on the right of the untreated spheres panel and 40 µm on the right of the Roniciclib treated spheres panel). ( B ) Average diameter of neurospheres produced by untreated IMR-32, ACN and SH-SY5Y cells (u) and by Roniciclib treated cells (R). Data are representative of three independent experiments ± SD (*** p < 0.001).

Article Snippet: Roniciclib (BAY 1000394), by AdooQ Bioscience (Irvine, CA, USA), was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM and maintained at − 20 °C until used.

Techniques: Produced, Cell Culture

Journal: Scientific Reports

Article Title: Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

doi: 10.1038/s41598-020-69499-6

Figure Lengend Snippet: Roniciclib strongly inhibits cancer stem cells markers expression while enhances p53 onco-suppressor level in neuroblastoma cells. ( A ) 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-CD44v6, anti-CD114, anti-Osteopontin (OPN), anti-Microtubule-associated protein-2 (MAP2), anti-p53, anti-β-catenin and anti-Low density lipoprotein related protein (LRP6) antibodies. Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Supplementary Figure , panels 1–2, shows the entire blots images. ( B ) 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), then visualized by histograms. Data are representative of three independent experiments ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Roniciclib (BAY 1000394), by AdooQ Bioscience (Irvine, CA, USA), was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM and maintained at − 20 °C until used.

Techniques: Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

doi: 10.1038/s41598-020-69499-6

Figure Lengend Snippet: 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 , 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).

Article Snippet: Roniciclib (BAY 1000394), by AdooQ Bioscience (Irvine, CA, USA), was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM and maintained at − 20 °C until used.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, TUNEL Assay, Staining, Generated

Journal: Scientific Reports

Article Title: Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

doi: 10.1038/s41598-020-69499-6

Figure Lengend Snippet: Roniciclib reduces tumor growth in a neuroblastoma xenograft model. 1 × 10 6 IMR-32 cells were injected in the capsule f the left adrenal gland of nude mice. Mice received oral administration of vehicle control (n = 10) or of Roniciclib (1.5 mg/kg once daily) (n = 10) upon tumor growth. ( A ) Curves indicate tumor volume with vehicle control or with Roniciclib treatment over 14 days. ( B ) In vivo characterization of orthotopic neuroblastoma after 14 days of control vehicle or Roniciclib treatment. For histological analysis, sections were stained with hematoxylin and eosin (H & E). Serial sections were treated by immunohistochemistry with anti-Ki-67 and anti-CD31 (brown) antibodies and by immunofluorescence with anti-MAP2, anti-CD44v6, anti-NCL, anti-NPM1 and PES1 (all green) antibodies. Cells nuclei are counterstained with hematoxylin (violet) or with DAPI (blue). ( C ) The histograms represent the percentage of Ki-67 + , CD31 + , MAP2 + , CD44v6 + , NCL + , NPM1 + and PES1 + cells. Data are representative of three independent observations ± SD (* p < 0.05; *** p < 0.001).

Article Snippet: Roniciclib (BAY 1000394), by AdooQ Bioscience (Irvine, CA, USA), was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 20 mM and maintained at − 20 °C until used.

Techniques: Injection, Control, In Vivo, Staining, Immunohistochemistry, Immunofluorescence