Journal: Nature communications

Article Title: The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma.

doi: 10.1038/s41467-021-26640-x

Figure Lengend Snippet: Fig. 3 CX-5461 causes DNA damage and cell death by apoptosis in neuroblastoma cells at sub-micromolar concentrations. a Apoptosis in CHP-134 cells. The cells were treated for 48 h. b Quantification of data in (a). Data represent the mean ± SD of three independent experiments. Significant P-values (left to right): 3.9 × 10−6, 7.2 × 10−7, and 4.8 × 10−7. c Quantification of flow cytometry data showing apoptosis up to 48 h in CHP-134 and IMR-5 (0.2 µM and 0.1 µM of CX-5461 respectively). CHP-134 cells were treated with 0.2 µM and IMR-5 cells with 0.1 µM of CX-5461. Data represent the mean ± SD of three independent experiments. Significant P-values (left to right): CHP-134, 2.2 × 10−4, 3.2 × 10−5, and 1.5 × 10−5; IMR-5, 1.8 × 10−4, 2.3 × 10−5, and 2.5 × 10−6. d Western blots for cleaved PARP (c-PARP). β-Actin was used as a loading control. Representative results of three biological replicates are shown. e Co-localization of 53BP1 and γ-H2AX. Cells were treated for 24 h. Representative results of three biological replicates are shown. Scale bar = 10 μm. See Fig. S3d for more time points. f Comet assay in CHP-134 cells. Tail length (left) and representative images (right) are shown. The red line indicates the mean value of tail length in each group. Scale bar = 5 µm. g Cell cycle analysis forCHP-134 (0.2 µM CX-5461) and IMR-5 (0.05 µM CX-5461) cells. Data represent the mean ± SD of three independent experiments. P-values vs 0 h: P1 = 4.2 × 10−5, P2 = 7.1 × 10−7, P3 = 6.3 × 10−5, P4 = 2.7 × 10−4, P5 = 6.3 × 10−3, P6 = 2.3 × 10−5, P7 = 1.8 × 10−4, and P8 = 0.044. h EdU incorporation assay to assess global nascent DNA transcription. Representative results of three biological replicates are shown. Blue, DAPI; green, EdU. Scale bar = 20 µm. i CX-5461 induced DNA damage (γ-H2AX, red) was enriched in cells with active DNA synthesis (positive for EdU (green) labeling). Representative results of three biological replicates are shown. Scale bar = 10 μm. Two-tailed t- test was used for (b, c, g). Source data for these panels are included in Source Data file.

Article Snippet: ProLong® Gold Antifade Reagent with DAPI (Cell Signaling, #8961) was used as coverslip mountant.

Techniques: Cytometry, Western Blot, Control, Single Cell Gel Electrophoresis, Cell Cycle Assay, DNA Synthesis, Labeling, Two Tailed Test