Shh induces aerobic glycolysis in CGNPs. (A) Counts of EdU+ cells, in 3 replicate wells for each condition, confirm that Shh-treated CGNPs continue proliferation after 48 hours in culture, while vehicle-treated CGNPs exit the cell cycle. (B) Lactate production, glucose uptake and oxygen consumption rate (OCR) of Shh-treated and vehicle-treated CGNPs are compared, using 3 replicate wells per condition. Measured values were normalized for cell number and expressed as fold-change relative to vehicle-treated values. Shh increased lactate production (P < 0.01) and glucose uptake (P < 0.03) while no statistically significant effect on the OCR was detected. (C) NMR spectra (representative examples on top; below is orthogonal partial least squares discriminant analysis comparison of 6 replicates of each condition) demonstrate differential accumulation of lactate in media of Shh-treated CGNPs compared with vehicle-treated CGNPs. The loading coefficient is plotted as the y value, and the P scaled correlation coefficient is color-coded as indicated. Lactate peaks are deflected toward Shh, indicating greater value in Shh-treated wells, and color-coded red, indicating statistical significance. (D) Starting with fresh media at time 0, Shh-treated CGNPs used more glucose (P < 0.001) and produced more lactate (P < 0.001) than vehicle-treated CGNPs over a 6-hour period. Importantly, in Shh-treated CGNPs, glucose utilization and lactate production were in a stoichiometric 1:2 ratio. Graphs present mean ± standard error of the mean (SEM). Two-tailed Student’s t test was used for statistical comparisons in (A) and (B), while two-way analysis of variance with Bonferroni correction was used in (D).