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. 2024 Nov 6;27(12):111336. doi: 10.1016/j.isci.2024.111336

Figure 7.

Figure 7

HYlight quantifies fructose 1,6-bisphosphate (FBP) concentrations in liver cells

(A) Changes in relative fluorescence ratio were observed as a function of FBP concentration in various buffers that simulate cellular ionic strength and metabolic composition. Sample size is n = 3.

(B) Relative fluorescence ratio change as a function of FBP concentration in HepG2 cells under saponin permeabilization and comparison with recombinant HYlight with CIS + metabolic mix. Sample size is n = 6.

(C) Comparison of FBP concentrations in HepG2 cells under glucose starvation, 10 mM glucose, 2.5 μM oligomycin, 50 mM 2-DG obtained via mass spectrometry (gray) and compared to normalized fluorescent ratios (ΔR/R10) of HYlight in HepG2 cells (blue), correlated with recombinant HYlight (open circles). Sample size of metabolomics analysis is n = 1 with 3 technical replicates.

(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, and oligomycin. FBP quantification via correlation of normalized fluorescent ratio of HYlight in HepG2 cells with normalized fluorescence ratio ΔR/R10 of recombinant HYlight. Scale bar, 100 μm.

(E) FBP quantification in mouse hepatocytes, Huh6 and HLE cell after 10 mM glucose addition via correlation of normalized fluorescent ratio of HYlight in HepG2 cells with normalized fluorescence ratio ΔR/R10 of recombinant HYlight. Sample size is n = 3 with a minimum of 50 cells per replicate. CIS: cytosolic ionic strength. 140 mM KCl, 12 mM NaCl; PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; TBS: 150 mM NaCl, 50 mM Tris-HCl; SEC (size exclusion chromatography) buffer: 50 mM Tris pH 7.5, 125 mM NaCl; metabolic mix: 4.67 mM ATP, 0.096 mM fructose 6-phosphate, 0.67 mM glucose 6-phosphate, 1.63 mM dihydroxyacetone phosphate, 0.14 mM glyceraldehyde 3-phosphate. ΔR/R10=(ratio 500/400 – ratio 500/40010 μM FBP)/ratio 500/40010 μM FBP where ratio 500/40010 μM FBP is the ratio with 10 μM FBP. Data are presented as the mean ± SD. See also Figure S5 and Table S1 for additional details. (C–E) can be also accessed through BioRender: BioRender.com/l93y325; BioRender.com/r87j960; BioRender.com/c21p293.