to monitor carbon flux in the transformed iNPCs as compared with WTiNPCs. No significant differences in levels of production of glycolytic metabolites were found among the four lines, except for increased glucose flux into lactate and glycine in p53KD-Ras/EGFR/SrciNPCs, (Supplementary Fig. 3d). Interestingly, we observed much lower influx of glucose into glutamate production and into the trichloroacetic acid or tricarboxylic acid cycle (as highlighted by differential labelling of aspartate, malate, fumarate and α-ketoglutarate) in Ras/EGFR/SrciNPCs and p53KD-Ras/EGFR/SrciNPCs, compared with WT- and p53KDiNPCs, which shared a similar metabolic profile (Supplementary Fig. 3d). Quantification of ROS levels further highlighted metabolic reprogramming and demonstrated reduced mitochondrial ROS formation upon iNPC transformation, whereas total ROS levels remained comparable across all different groups (Supplementary Fig. 4a,b). MitoTracker analysis indicated that disruption of signalling pathways resulted in morphological mitochondrial changes and higher fragmentation of mitochondrial networks as compared with WTiNPCs (Supplementary Fig. 4c).
