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Figure 6 | BMC Immunology

Figure 6

From: Cytokine generation, promoter activation, and oxidant-independent NF-κB activation in a transfectable human neutrophilic cellular model

Figure 6

Transient transfection of neutrophil-like PLB-985 cells with promoter-reporter constructs. (A) Granulocytic PLB-985 cells (day 5) were nucleofected with luciferase constructs driven by 5 repeated NF-κB elements ("pNFkB-Luc"), or by 7 repeats of the consensus AP-1 element ("pAP1-Luc"). The cells were then cultured for 6 h at 37°C in the presence or absence of 100 ng/ml LPS or 100 U/ml TNFα, prior to cell lysis and luciferase activity measurement. Mean ± s.e.m. of at least 3 independent experiments. Transfections made using empty vectors yielded values that did not differ significantly from background, whether in resting or stimulated cells (not shown). (B) Granulocytic PLB-985 cells (day 5) were nucleofected with a luciferase construct driven by the IL-8 promoter ("pIL-8-Luc"), and cultured for 6 h at 37°C in the presence or absence of 100 ng/ml LPS or 100 U/ml TNFα, prior to cell lysis and luciferase activity measurement. Mean ± s.e.m. from 8 independent experiments. (C) Granulocytic PLB-985 cells (day 5) were nucleofected with luciferase constructs encoding either either a wild-type ("wt") IL-8 promoter or a variant mutated in the NF-κB site ("mut κB"). The cells were then cultured for 6 h at 37°C in the presence or absence of 100 U/ml TNFα, prior to cell lysis and luciferase activity measurement. Mean ± s.e.m. from 2 independent experiments.

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