Figure 1.

HYlight monitors fructose 1,6-bisphosphate (FBP) in hepatocytes and liver cancer cells

(A) Min-max normalized ratio changes of HYlight in primary mouse hepatocytes after 1 h of glucose starvation upon sequential addition of increasing concentrations of glucose every 10 min and 2-deoxy-D-glucose (2-DG) at the end of the experiment. Data are presented as mean ± SEM. Averaged fluorescence ratio upon compound addition. Mean ± SD. Single-cell fluorescence ratio upon compound addition. n = 3 and at least 10 cells per replicate.

(B) Fluorescence ratio changes of codon-optimized HYlight in HLE, Huh6, and HepG2 cell lines as well as primary mouse hepatocytes following 1 h of glucose starvation. This was followed by the sequential addition of increasing concentrations of glucose every 10 min, and subsequent addition of 2-deoxy-D-glucose (2-DG). Data are presented as mean ± SEM. n = 3 and at least 50 cells per replicate.

(C) The HYlight fluorescence ratio as a function of glucose concentration exhibits a hyperbolic response curve in HLE, HepG2, and Huh6 cell lines, as well as primary mouse hepatocytes. n = 3 and at least 50 cells per replicate.

(D) Fluorescence ratiometric images (fusion of 488 and 405 nm wavelength) of HepG2 cells after 1 h of glucose starvation and after the addition of glucose, oligomycin, and 2-DG. Scale bar, 100 μm. Inner scale bar, 50 μm. Below, the normalized min-max ratiometric change and the averaged ratio change of HYlight in HepG2 cells under physiological stimuli. n = 3 and at least 50 cells per replicate. Data on the left graph are presented as mean ± SEM and on the right graph as mean ± SD. Scale bar: 200 μm ns: Not significant; ∗<0.05; ∗∗<0.005; ∗∗∗<0.0005; ∗∗∗∗<0.00005. Ordinary One-Way ANOVA Šídák's Multiple Comparisons Test. See also data S1 and Figure S1 for additional details.