Article Figures & Data
Figures
- Figure 1.
The schematic presentation of the molecular basis of targeting PFKFB3 as a therapeutic strategy against melanoma (A) (Adapted from Ref. 19) and (B) the 2D structure of 3PO (20). EGR: Epidermal growth factor; EGFR: epidermal growth factor receptor; HIF-1α: hypoxia-inducible factor α; Fru-2,6-BP: fructose 2,6-bisphosphate; F-1,6-BP: fructose 1,6-bisphosphate; PFK-1: phosphofructokinase-1; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3; DHA: dihydroxyacetone: G3P: glyceraldehyde 3-phosphate.
- Figure 2.
Simulation of the 3PO binding efficacy: A) docking of the 3PO ligand using the Smina/Vinardo (red) and docking of the native ligand F-2,6-BP using the Autodock 4 (green). The 3PO binds to the 2-Pase domain, same as the F-2,6-BP native ligand, indicating why it inhibits the PFKFB3. The 2-Pase domain is shown in the cyan color, where the 2-Kase domain is shown in brick color. B) Amino acids within 3Å of the 3PO docked ligand. The number near the amino acid's name indicates a position in the protein structure. The two amino acids at the back of the image are glutamine 322 and isoleucine 323. C) Amino acids, that are shared between the docked 3PO ligand and the docked F-2,6-BP native ligand. The amino acids are shown within 3Å of both ligands.
- Figure 3.
Cytotoxic effect of 3PO on A375 melanoma cells: A) Viability of A375 cells after 24 and 48 h of incubation with 3PO measured using the MTT assay. B) Morphology of the A375 melanoma cells incubated in medium without (Ctrl) or with 3PO at a concentration of 5, 25 and 100 μM. C) Morphology of the A375 melanoma cell line incubated in medium alone (Ctrl) or medium with 3PO at a concentration of 5, 25 and 100 μM. The pictures and 3D representations were acquired using the holotomography microscope (3D Cell Explorer, Nanolive). *p<0.05. Scale bars: 200 μm. INH: Inhibition.
- Figure 4.
Cell death evaluation of A375 cells after 24 h of incubation without 3PO (Ctrl) and with 3PO at the concentration of 5, 25 and 100 μM (staining with Annexin-FITC and PI).
- Figure 5.
Apoptosis intensity after 24h incubation with 3PO. The representative microscopy images present cytoplasmatic expression of caspase 3 and 8 in A375 melanoma cells. The IHC reaction is stronger in cells exposed to the higher 3PO concentrations. Scale bars: 100 μM.
- Figure 6.
The glucose uptake in A375 cell line after 24 h of incubation with 3PO. *p<0.05, **p<0.01, ****p<0.0001 compared to control. RLU: Relative luminescence units.
- Figure 7.
Clone formation efficacy in A375 cell line after 24 h of incubation with 3PO. **p<0.01 compared to control.
- Figure 8.
Cell cycle analysis of A375 cells presented as histograms of PI intensity staining after 24 h of incubation with different 3PO concentrations and its summary (bottom right graph).
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