Fig. 5: Expression of MYCN in ATRX-mutant cells leads to ROS production, DNA damage, and replicative stress.
From: MYCN amplification and ATRX mutations are incompatible in neuroblastoma
a Bar plot (mean±SD) of ROS levels on Day 6 ± doxycycline. P value was calculated using two-tailed Student t test. b Immunoblots on Day 4 ± doxycycline. The experiment was repeated with the same results. c Immunofluorescent detection of γH2AX (red) and nuclei (blue) in SKNMMMYCN cells on Day 4 ± doxycycline. The experiment was repeated with the same results. d Spectral karyotype analysis (SKY) of SKNMMMYCN cells ± DOX on Day 8. Chromosomes are shown adjacent to the pseudo-colored representation. Arrows indicate translocations. The pie charts show the proportion of cells with DNA fragmentation (n = 50). P value was calculated using Chi-square test. e, f Micrographs of single-cell electrophoresis of individual nuclei (dashed circles) and their COMET tail (arrows) ± doxycycline on Day 5. g Mean±SD of the COMET assay scoring. The number of analyzed cells are presented on the graph. h Photograph of cresyl violet-stained colonies from SKNMMMYCN cells ± doxycycline, with increasing concentrations of retinoic acid (RA). i Immunoblots of SKNMMMYCN cells ± doxycycline, with or without 5 μm RA. The level of MYCN protein was reduced by 30% (0.7×) in the presence of RA. j ChromHMM and ChIP-seq for MYCN, H3K4me3, and H3K27me3 for the CUX2 promoter for a MYCN-amplified neuroblastoma (SJNBL012407_X1) xenograft and SKNMMMYCN cells in the presence or absence of doxycycline after 4 days in culture. The gene expression (FPKM) is indicated, and the dashed line indicates the start of transcription. k Line plot of SKNMMMYCN cells in the presence or absence of doxycycline after 4, 6, 8, and 10 days in culture with or without ectopic expression of CUX2 or the GFP control from lentiviral infection. Each point is the mean and standard deviation of triplicate experiments, and the asterisks indicate statistical significance (P = 0.005, 0.001, and 0.008 at days 6, 8, and 10, respectively, two-tailed Student's t test). Scale bars e, f, 10 μm. DOX doxycycline, RA retinoic acid, ROS reactive-oxygen species, m marker chromosome fragments that could not be definitively identified.
