3 3 pyridinyl 1 4 pyridinyl 2 propen 1 one 3po

The commercial PFKFB3 inhibitor, 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) was purchased from Merck Millipore. Cells were cultured in the medium added 3PO (10 μM) as the experimental group, and the control group was absence of 3PO. After incubation for 24 h, glycolysis flux (glucose uptake and lactate secretion analysis) and cell viability (CCK8 assay) were detected. The experiments were carried out in triplicate.

3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) was purchased from EMD Millipore (MA, USA). Pinocembrin and other reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA).

2×10⁶ human or mouse fibroblasts were seeded in 30 mm dishes and cultured overnight. To test the factors that would affect the uptake of IR-780, cells were treated with different factors: 1) aerobic glycolysis: Cells were treated with 2-Deoxy-D-glucose (2-DG, 150mM) for 45 min or 6-aminonicotinamide (6-AN,5μm,Sigma) for 24 hours or 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO,10 μM, Sigma) for 24 hours respectively. 2) organic-anion-transporting polypeptides (OATPs): Cells were treated with sulfobromophthalein disodium salt hydrate (BSP, 250μM, Sigma) for 5min or SLCO2A1, SLCO1B3 siRNA for 48 hours. 3) HIF-1a pathway: Cells were treated with CTGF siRNA for 48 hours or 5% hypoxia for 48 hours or 5% hypoxia+LW6 (a HIF-1α inhibitor, 10μM, Selleck) for 48 hours. 4) endocytosis: Cells were treated with amiloride(50μg/mL) or chlorpromazine (3.75μg/mL) or methyl-β-cyclodextrin (4×10^-3M ) for 1 hour respectively. Cells were then incubated with 1μM IR-780 in DMEM for 20 min at 37°C. Following thrice washes with PBS, NIR images were directly captured by fluorescent microscope (Leica, excitation/emission: 770/830). Or cells were fixed by 4% polyoxymethylene for 15 min at RT and stained with DAPI for 1 min. NIR images were then captured.

Inhibitors were added to neutrophils at the following final concentrations 1 hr prior to infection, unless otherwise indicated: 20 µM WZB-117 (20 mM stock in DMSO, Sigma-Aldrich), 5 mM 2-deoxy-D-glucose (2-DG) (100 mM stock in RPMI-1640, Sigma-Aldrich), 50-100 μM 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO, 100 mM stock in DMSO, Sigma-Aldrich), 20 µM CP-91149 (20 mM stock in DMSO, Sigma-Aldrich), or 40 µg/ml chlorogenic acid (40 mg/ml stock in DMSO, Sigma-Aldrich). Q-VD-OPh (10 mM stock in DMSO, Cayman Chemical) was added to a final concentration of 10 µM, at time zero simultaneously with initiation of infection (no pretreatment).