Fig. 3: Shikonin induces ATP depletion due to inhibition of glycolysis in PDAC cells.

a ATP was measured using a 96-well plate based on firefly luciferase luminescence assay in Mia PaCa-2 cells. Cells were treated with different concentrations of PKM2 activator, TEPP-46 (10–60 µM, grey bars), PKM2 inhibitor and shikonin (0.1–10 µM, black bars). DMSO and methanol were used as vehicle controls (white bars), and 2 mM iodoacetate (IAA, hash bars) and 10 µM oligomycin (OM, black bars) were used as positive and negative controls, respectively. ATP luminescence was measured at 15 min (ai), 1 h (aii), 6 h (aiii) and 24 h (aiv) post drug treatment. Raw luminescence counts were normalised to an identical corresponding plate stained with SRB prior to normalising to vehicle controls. Significance was determined using a Kruskal–Wallis test with Dunn’s test (*p < 0.05). Data were averaged across multiple replicates (four per experiment) for four experiments. b Simultaneous measurement of oxygen consumption rate (OCR, mitochondrial metabolism) and extracellular acidification rate (ECAR, glycolysis), using the Seahorse XF Analyzer, and the quasi-simultaneous, time-matched measurement of cytosolic ATP using GO-ATeam FRET microscopy. Data were normalised to the third control measurement (10 min) prior to addition of 10 µM oligomycin (OM) to inhibit mitochondrial F₁F₀-ATP synthase (bi) or 2 mM iodoacetate (IAA), to inhibit the glycolytic enzyme GAPDH (bii). c Concentration-dependent effect of shikonin (1–10 µM) on normalised ECAR (% of control) in Mia PaCa-2 cells cultured in high- (ci, 25 mM) or low- (cii, 5 mM) glucose DMEM.