Fig. 2: The MYC-NOX4 axis promotes ROS generation and nucleotide oxidation.

a Time course analysis of ROS levels in 4-OHT (200 nM) treated SHEP MYCN-ER cells. One million cells were stained with DCFH-DA (5 µM) for 20 min at 37 °C, followed by detection with flow cytometry. b Real-time qPCR analysis and immunoblot of NOX4 in SHEP MYCN-ER cells upon 4-OHT treatment (200 nM). c ChIP-qPCR analysis of MYCN binding to the NOX4 promoter in SHEP MYCN-ER cells. d Detection of ROS levels in SHEP MYCN-ER cells transduced with the NOX4 sgRNA and treated with 4-OHT (200 nM) for 48 h. e Representative avidin immunofluorescence images in SHEP MYCN-ER cells. Cells were transduced with the NOX4 sgRNA and treated with 4-OHT (200 nM) in the presence or absence of NAC (100 µM), or OGG1 overexpression. Scale bar, 20 μm. f Quantification of 8-oxo-dGTP incorporation in SHEP MYCN-ER cells (n = 10 images) as shown in (e). Data are means ± SD. g Cell death analysis of SHEP MYCN-ER cells treated as shown in (e). h Representative avidin immunofluorescence images in Kelly cells transduced with the NUDT1 shRNA and NOX4 sgRNA lentivirus particles. Scale bar, 20 μm. Quantification of immunofluorescence signals are shown on the right (n = 10 images). Data are means ± SD. i Death analysis of Kelly cells transduced with the NUDT1 shRNA and NOX4 sgRNA lentivirus particles. b, c, g, i data are shown as averages of technical triplicates, and these experiments were independently repeated three times with similar results; e, h these experiments were independently repeated twice with similar results. Statistical significance was determined by one-way ANOVA (b) or two-way ANOVA (c, f–i). Source data are provided as a Source Data file.