Figure 5.
Figure 5. Activation of TCR leads to CXCR4-mediated stabilization of cytokine mRNA by activation of a PREX1-Rac1–signaling pathway. (A-D) PBMC T cells were treated with 60 μM AMD3100 or 50 μM NSC23766 for 1 hour, stimulated with 5 μg/mL biotinylated OKT3 crosslinked with avidin and soluble CD28 for 5 minutes, and assayed for active, GTP-bound Rac1, or total Rac1. Vertical white lines between bands indicate removal of an irrelevant lane from the gel image. (B,D) Summary of results as in panels A and C, respectively; each bar denotes the fold increase in activated Rac1 in stimulated cells compared with unstimulated cells, (n = 3-5; P < .05). (E) PBMC T cells were treated with 50 μM NSC23766 for 1 hour, stimulated with 1 μg/mL plate-bound OKT3 and soluble CD28 for 5.5 hours prior to addition of actinomycin D and assessment of mRNA levels as in Figure 4I (n = 7-10; P < .05). Donor samples were included in this analysis only if there was less than a twofold difference in starting transcript levels between treated and untreated samples at 1 minute of actinomycin D treatment. (F) PBMC T cells were transfected with pmir-GLO empty vector or pmir-GLO-3′UTR at 350 V on a BTX square wave electroporator, treated with 60 μM AMD3100 or 50 μM NSC23766 for 1 hour, stimulated with 1 μg/mL plate-bound OKT3 and soluble CD28 Ab for 5.5 hours, harvested, lysed, and assayed for luciferase activity. Luciferase activity of pmir-GLO-3′UTR was normalized to pmir-GLO empty vector. Representative experiment is shown with each bar denoting mean relative light units ± SD (n = 3; P < .05) (unpaired t test). (G) PBMC T cells were treated with 50 μM NSC23766 for 1 hour, stimulated as in panel F for 24 hours, and supernatants were analyzed for the indicated cytokines as in Figure 1D-E (n = 6). (H-M) PBMC T cells were transfected with PREX1 siRNA-1 (pool of siRNAs), PREX1 siRNA-2 (single siRNA) or control siRNA, cultured for 48 hours, and stimulated as indicated below. (H-I,K-L) Cells were stimulated as in panel A and harvested to either immunoblot PREX1 and actin expression or assay GTP-bound Rac activation as in panels A and B (n = 3). (J,M) Forty-eight hours after transfection, cells were stimulated as in panel G for 24 hours and supernatants were analyzed for the indicated cytokines as in Figure 1D-E (n = 6).

Activation of TCR leads to CXCR4-mediated stabilization of cytokine mRNA by activation of a PREX1-Rac1–signaling pathway. (A-D) PBMC T cells were treated with 60 μM AMD3100 or 50 μM NSC23766 for 1 hour, stimulated with 5 μg/mL biotinylated OKT3 crosslinked with avidin and soluble CD28 for 5 minutes, and assayed for active, GTP-bound Rac1, or total Rac1. Vertical white lines between bands indicate removal of an irrelevant lane from the gel image. (B,D) Summary of results as in panels A and C, respectively; each bar denotes the fold increase in activated Rac1 in stimulated cells compared with unstimulated cells, (n = 3-5; P < .05). (E) PBMC T cells were treated with 50 μM NSC23766 for 1 hour, stimulated with 1 μg/mL plate-bound OKT3 and soluble CD28 for 5.5 hours prior to addition of actinomycin D and assessment of mRNA levels as in Figure 4I (n = 7-10; P < .05). Donor samples were included in this analysis only if there was less than a twofold difference in starting transcript levels between treated and untreated samples at 1 minute of actinomycin D treatment. (F) PBMC T cells were transfected with pmir-GLO empty vector or pmir-GLO-3′UTR at 350 V on a BTX square wave electroporator, treated with 60 μM AMD3100 or 50 μM NSC23766 for 1 hour, stimulated with 1 μg/mL plate-bound OKT3 and soluble CD28 Ab for 5.5 hours, harvested, lysed, and assayed for luciferase activity. Luciferase activity of pmir-GLO-3′UTR was normalized to pmir-GLO empty vector. Representative experiment is shown with each bar denoting mean relative light units ± SD (n = 3; P < .05) (unpaired t test). (G) PBMC T cells were treated with 50 μM NSC23766 for 1 hour, stimulated as in panel F for 24 hours, and supernatants were analyzed for the indicated cytokines as in Figure 1D-E (n = 6). (H-M) PBMC T cells were transfected with PREX1 siRNA-1 (pool of siRNAs), PREX1 siRNA-2 (single siRNA) or control siRNA, cultured for 48 hours, and stimulated as indicated below. (H-I,K-L) Cells were stimulated as in panel A and harvested to either immunoblot PREX1 and actin expression or assay GTP-bound Rac activation as in panels A and B (n = 3). (J,M) Forty-eight hours after transfection, cells were stimulated as in panel G for 24 hours and supernatants were analyzed for the indicated cytokines as in Figure 1D-E (n = 6).

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