Figure 3

Vitamin D restrained anaerobic glycolysis. HEK293T cells were treated for 24 hours with 100 nM of 1,25 dihydroxyvitamin D3, after cell medium was changed by the same medium except ¹³C-U-glucose. Cell medium was aliquoted every 15 minutes, up to 180 minutes. After 30 minutes, antimycin A (inhibitor of mitochondrial complex III) was added. Lactate in the culture medium was measured by one-dimension ¹³C-NMR spectra acquisition, with proton decoupling, of aliquots of the culture medium. ¹³C-Lactate accumulation was calculated by the ratio of peak intensities in 180 min:30 min. We measured the lactate peak intensity for every 15 min. The intensity ratio 180/30 min normalizes for small changes at the beginning of the kinetics (30 min) and better reports for the significance of the effect of vitamin D and antimycin. Only after 120 min (we chose 180 min) the difference among the tested condition arose. Values represent the average of 3 independent experiments, ± SD. For the analysis of the statistical significance with and without vitamin D we used unpaired t-test (*p < 0.05).