Fig. 4: Expression of MYCN in ATRX-mutant cells leads to metabolic reprogramming and mitochondrial dysfunction.

a Simplified drawing of the TCA cycle and reductive carboxylation, blue circles: ¹³C derived from ¹³C₅-glutamine. b Bar plot (mean of two technical replicates) of ¹³C-labeled isotopomers in SKNMM^MYCN cells on day 4 ± doxycycline after 5 h of labeling with ¹³C₅-glutamine. The arrows indicate an increase in M + 5 glutamate and M + 5 α-ketoglutarate (the most abundant and direct derivatives of ¹³C₅-glutamine) in the presence of doxycycline. c Photograph of culture media on day 3 ± doxycycline. d Line plots of media pH ± doxycycline for each day. The culture media were changed on Day 3. e Histogram (mean of two biological replicates) of the levels of lactate at Day 4 in SKNMM^MYCN cells ± doxycycline. f Simplified drawing of the glycolysis highlighting the production of lactate from ¹³C₆-glucose. g Bar plots of ¹³C-labeled isotopomers of lactate and α-ketoglutarate in the medium or cells after 18 h of labeling with ¹³C₆-glucose or ¹³C₅-glutamine, respectively, at Day 4 in SKNMM^MYCN cells ± doxycycline, with concentrations indicated on the bars. h, i Box and Whiskers plots of MitoTracker Green (H) and TMRE (I) staining ± doxycycline on Day 4. Each Box shows the 10th to 90th percentiles range of data (at least 15,000 cells), the line represents the median and the Whiskers show the minimum and maximum data range. P values were calculated using two-tailed Mann–Whitney non-parametric test. j, k Representative results of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (mean and SEM of three technical replicates) for 2.5 × 10⁴ SKNMM^MYCN cells grown under the Mito-Stress assay conditions for the Seahorse. The experiment was done three times with similar results. l–o Representative electron micrograph of mitochondria from SKNMM^MYCN cells after 4 days ± doxycycline l, n showing disruption in the mitochondrial cristae (arrows). Bar plot of scoring of mitochondrial morphology for the SKNMM^MYCN cells ± doxycycline, numbers of scored mitochondria are indicated m, o. P value was calculated using Chi-square test. DOX doxycycline, FCCP carbonyl cyanide-p-trifluoromethoxy phenylhydrazone, Olig oligomycin, Rot/AA rotenone/antimycin A, TCA tricarboxylic acid, TMRE tetramethylrhodamine ethyl ester.