American Society of Hematology

Figure 4.

$Figure 4. AMD3100 inhibits cytokine mRNA stability without altering TCR-initiated ERK-, NF-κB–, or NFAT-signaling pathways. (A-G) PBMC T cells were pretreated with AMD3100 for 1 hour and stimulated as indicated. (A-D) T cells were stimulated with 1 μg/mL biotinylated OKT3 crosslinked with avidin and soluble CD28 for the indicated time. (A) Representative results of ERK activation assayed by flow cytometry. (B) Summary of results in panel A; each bar denotes the mean fold increase of ERK activation over unstimulated cells, ± SEM (n = 4). (C) Representative results of cellular lysates that were isolated and blotted for IκBα degradation. (D) Summary of results in panel C; each bar denotes the mean percentage of IκBα remaining after stimulation compared with unstimulated cells (n = 3). (E-I) Cells were stimulated with CD3 + CD28 as in Figure 1. (E) Representative results of subcellular fractions isolated after 6 hours of CD3 + CD28 stimulation and immunoblotted for NFATc1. (F) Summary of panel E; each bar denotes mean fold increase in NFAT nuclear localization after stimulation compared with unstimulated cells (n = 3). (G) After stimulation with CD3 + CD28 for the indicated time, the cells were harvested for analysis via qRT-PCR for the indicated mRNA transcript levels. The results shown are normalized to the reference gene GAPDH, where GAPDH is set to 100. Each point denotes the mean mRNA transcript level ± SEM for 4 independent donors. (H) Jurkat T cells were transfected with an IL-2 promoter luciferase reporter construct, incubated for 16 to 18 hours, treated with 60 μM AMD3100 for 1 hour, stimulated as in Figure 1 for 16 hours, and assayed for luciferase activity. Representative experiment is shown. Each bar denotes the mean relative light units, ± SD (n = 4; P > .05) (unpaired Student t test). (I) PBMC T cells were pretreated with AMD3100 for 1 hour and stimulated as in Figure 1 for 5.5 hours prior to addition of actinomycin D. mRNA levels were assayed via qRT-PCR following actinomycin D treatment for the indicated times. Each point denotes the mean percentage of mRNA remaining ± SEM. *Significantly different from control cells (n = 3-4). C, cytoplasmic; N, nuclear; N.S., no significant difference (P > .05).$

AMD3100 inhibits cytokine mRNA stability without altering TCR-initiated ERK-, NF-κB–, or NFAT-signaling pathways. (A-G) PBMC T cells were pretreated with AMD3100 for 1 hour and stimulated as indicated. (A-D) T cells were stimulated with 1 μg/mL biotinylated OKT3 crosslinked with avidin and soluble CD28 for the indicated time. (A) Representative results of ERK activation assayed by flow cytometry. (B) Summary of results in panel A; each bar denotes the mean fold increase of ERK activation over unstimulated cells, ± SEM (n = 4). (C) Representative results of cellular lysates that were isolated and blotted for IκBα degradation. (D) Summary of results in panel C; each bar denotes the mean percentage of IκBα remaining after stimulation compared with unstimulated cells (n = 3). (E-I) Cells were stimulated with CD3 + CD28 as in Figure 1. (E) Representative results of subcellular fractions isolated after 6 hours of CD3 + CD28 stimulation and immunoblotted for NFATc1. (F) Summary of panel E; each bar denotes mean fold increase in NFAT nuclear localization after stimulation compared with unstimulated cells (n = 3). (G) After stimulation with CD3 + CD28 for the indicated time, the cells were harvested for analysis via qRT-PCR for the indicated mRNA transcript levels. The results shown are normalized to the reference gene GAPDH, where GAPDH is set to 100. Each point denotes the mean mRNA transcript level ± SEM for 4 independent donors. (H) Jurkat T cells were transfected with an IL-2 promoter luciferase reporter construct, incubated for 16 to 18 hours, treated with 60 μM AMD3100 for 1 hour, stimulated as in Figure 1 for 16 hours, and assayed for luciferase activity. Representative experiment is shown. Each bar denotes the mean relative light units, ± SD (n = 4; P > .05) (unpaired Student t test). (I) PBMC T cells were pretreated with AMD3100 for 1 hour and stimulated as in Figure 1 for 5.5 hours prior to addition of actinomycin D. mRNA levels were assayed via qRT-PCR following actinomycin D treatment for the indicated times. Each point denotes the mean percentage of mRNA remaining ± SEM. *Significantly different from control cells (n = 3-4). C, cytoplasmic; N, nuclear; N.S., no significant difference (P > .05).

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