Article Figures & Data
Figures
- FIGURE 1.
Perforin gene regulation in primary NK cells. Purified murine NK cells were stimulated with 100 U/ml IL-2 for 4 h in the presence or absence of 100 nM PDTC or 20 μM TPCK. RNA was extracted and analyzed by RT-PCR for perforin and cyclophilin A as an internal standard.
- FIGURE 2.
Regulation of perforin expression in NK3.3 cells stimulated with IL-2 in the presence or absence of 50 μM PDTC or 40 μM TPCK. A, The cell viability was assessed by the annexin V and PI staining after 5 h. B, RT-PCR analysis of perforin and β2m mRNA levels after 5 h. C, Nuclear run-on analysis of the perforin gene locus and β2m gene locus after 2 h of stimulation.
- FIGURE 3.
IL-2 stimulation of NK3.3 cells activates an NF-κB signaling pathway. A, Kinase assay analysis for IL-2-induced IKKα activity. NK3.3 cells were stimulated for the indicated times with IL-2, and IKKα immunoprecipitates were applied to in vitro kinase assays using rIκBα as substrate. The shown autorad is representative of two different experiments. B, IL-2-induced degradation of IκBα as determined by Western blot analysis of NK3.3 cells stimulated with IL-2. The result shown is representative of three independent experiments. C, EMSA of NF-κB DNA binding activity induced by IL-2 in NK3.3 cell nuclear extracts. NK3.3 cells treated with IL-2 for 4 h in the presence or absence of either 50 μM PDTC or 40 μM TPCK. The gel shift shown is representative of two different experiments. D, DNA binding specificities and identities of the IL-2-induced NF-κB-like binding activity. Cold competition EMSA and supershift assays with indicated probes and Abs were used to investigate the IL-2-induced NF-κB binding complex. The result shown is representative of three independent experiments.
- FIGURE 4.
NF-κB p50 binds to the upstream enhancer of the perforin gene. A, EMSA of the full-length perforin upstream enhancer and human recombinant p50 NF-κB. The binding reactions were conducted in the presence of the indicated amounts of NF-κB and the radiolabeled probe in the presence or absence of the indicated cold competitors. B, In vitro DNase I footprinting analysis of the perforin upstream enhancer for NF-κB p50 binding sites. A 32P 5′ end-labeled 117-bp DNA fragment containing the human perforin upstream enhancer was incubated with human recombinant p50, and subjected to DNase I footprinting analysis. C, Summary of the footprinting analysis of B. The sequence of upstream enhancer, three footprints of B (I, II, and III), three 2-bp mutations analyzed in Fig. 5, and the previously characterized tandem STAT element (10 ) are indicated. Probe I depicts the sequence used for EMSA in Fig. 6.
- FIGURE 5.
An NF-κB-like element of the perforin upstream enhancer is required for full enhancer activation by IL-2R signaling. A, Luciferase activity of the perforin upstream enhancer and three mutants (see Fig. 4) within the context of the SV40 promoter was determined upon cotransfection with pRL-CMV and standardization according to its activity in presence or absence of 500 IU/ml IL-2 in NK3.3 cells (n = 3). The first two bars represent the activity of the promotorless pGL3 vector in the absence and presence of IL-2. B, IL-2 induction (500 IU/ml) of the perforin upstream enhancer and the promotorless pGL3 vector in YT cells. The YT cells were transfected and analyzed as described for A (n = 3). The first two bars represent the activity of the promotorless pGL3 vector in the absence and presence of IL-2. C, Luciferase activity of the perforin upstream enhancer and mutant I (see Fig. 4) within the context of the SV40 promoter was determined upon cotransfection with pRL-CMV and standardization according to its activity in presence or absence of 500 IU/ml IL-2 in YT cells (n = 3).
- FIGURE 6.
IL-2-dependent and PDTC and TPCK sensitive NF-κB binding to the NF-κB consensus element of the perforin upstream enhancer (EMSA). Left panel, Effects of IL-2, PDTC, and TPCK on the binding activity of NK3.3 cell nuclear extracts to the perforin NF-κB element. Middle panel, Binding specificity of the IL-2-induced complex. The indicated wild-type or mutant NF-κB elements are used as probe. Right panel, The IL-2 induced perforin NF-κB consensus binding activity is specific in cold competition analyses.
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