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. 2024 May 27;115(8):2673–2685. doi: 10.1111/cas.16204

FIGURE 4.

FIGURE 4

Hypoxia promotes NF‐κB activation dependent on a HK2‐mediated IκBα T291 phosphorylation. (A, D, E) Immunoblot analyses were performed with the indicated antibodies. (A, B) PANC‐1 and AsPC‐1 cells with or without HK2 depletion were cultured for 24 h under normoxia or hypoxia. Cytosolic and nuclear fractions of the indicated cells were prepared (A). PANC‐1 and AsPC‐1 cells with or without HK2 depletion were stimulated with or without hypoxia for 24 h. Immunofluorescence analyses were performed with an anti‐p65 antibody (B). Scale bars, 10 μm. (C) PANC‐1 cells with or without HK2 depletion were cultured in the absence or presence of 3‐bromopyruvate (3‐BP) (25 μM). The indicated cells were transfected with luciferase reporter plasmid (NF‐κB‐Luc) and stimulated with or without hypoxia for 12 h. The data are presented as the mean ± SD of triplicate samples. N.S., not significant for the indicated comparison; ***p < 0.001. (D) PANC‐1 and AsPC‐1 cells with depleted HK2 and reconstituted expression of WT rHK2, rHK2 D209/D657A, or KD were cultured with or without hypoxia for 24 h. Cytosolic and nuclear fractions of the indicated cells were prepared. (E, F) PANC‐1 cells with depleted HK2 and reconstituted expression of WT rHK2, rHK2 D209/D657A, or KD were cultured with or without hypoxia for 24 h. Cytosolic and nuclear fractions of the indicated cells were prepared (E). Immunofluorescence analyses were performed with an anti‐p65 antibody. Scale bars, 10 μm. (G) WT rHK2, rHK2 D209/D657A, or KD was expressed in PANC‐1 cells with the depletion of HK2. The indicated cells were transfected with luciferase reporter plasmid (NF‐κB‐Luc) and stimulated with or without hypoxia for 12 h. The data are presented as the mean ± SD of triplicate samples. N.S., not significant for the indicated comparison; **p < 0.01; ***p < 0.001.