Figure 3
Figure 3. JAK/STAT5 signaling mediates B-cell defect in the absence of COX-1. (A) The expression of the indicated transcription factors in purified pro-B and pre-B cells from COX-1−/− and WT BM was determined by qRT-PCR. Mean ± SEMs from 4 independent experiments are shown. (B) Ebf1 and Pax5 expression was further confirmed by western blotting (WB). (Upper) From single representative experiments using mixed samples from 5 mice. (Lower) Mean ± SEMs from 3 independent experiments. (C) mRNA expression of Pax5 and Ebf1 in the distinct B-cell subpopulations was examined by qRT-PCR. We used β-actin for normalization, and the group with lowest expression was artificially set as 1. Data are presented as mean ± SEM from 3 independent experiments. (D) Purified B-cell subpopulations from mouse BM were stimulated with IL-7 for 15 min. The phosphorylation of STAT5 was analyzed by flow cytometry. (Left) Representative data from a single experiment. Solid, unstimulated; dotted, IL-7 stimulated. (Right) Mean ± SEMs from 3 independent experiments. MFI, mean fluorescence intensity. (E) Purified B-cell subpopulations were stimulated with IL-7 for 24 hours, and expression of STAT5 target genes were determined by qRT-PCR. Results represent mean ± SEM of 3 independent experiments. (F) BM cells were pretreated with the JAK3 inhibitor (WHI-P131, 15 μM) or dimethylsulfoxide (DMSO) for 12 hours, followed by administration of COX-1 inhibitor (SC-560, 20 μM). Cells were cultured under conditions to induce B-cell differentiation for 6 days. The percentages of pre-B cells (IgM−CD43−B220+) were analyzed by flow cytometry. Numbers indicate the frequencies of total living cells. (G-H) BM cells from COX-1−/− and WT mice were transduced with lentiviral plasmid (with GFP tag) expressing (G) constitutively activated STAT5 or (H) Pax5, and empty vector CPPT was used as the control. Infected cells were cultured under conditions for 6 days to induce B-cell differentiation. The percentages of total B cells and pre-B cells among GFP+ cells were evaluated by flow cytometric analysis. (F-H) (Left) Representative from a single experiment. (Right) Mean ± SEMs from 3 independent experiments. *P < .05 and **P < .01 using unpaired Student t tests and 1-way ANOVA.

JAK/STAT5 signaling mediates B-cell defect in the absence of COX-1. (A) The expression of the indicated transcription factors in purified pro-B and pre-B cells from COX-1−/− and WT BM was determined by qRT-PCR. Mean ± SEMs from 4 independent experiments are shown. (B) Ebf1 and Pax5 expression was further confirmed by western blotting (WB). (Upper) From single representative experiments using mixed samples from 5 mice. (Lower) Mean ± SEMs from 3 independent experiments. (C) mRNA expression of Pax5 and Ebf1 in the distinct B-cell subpopulations was examined by qRT-PCR. We used β-actin for normalization, and the group with lowest expression was artificially set as 1. Data are presented as mean ± SEM from 3 independent experiments. (D) Purified B-cell subpopulations from mouse BM were stimulated with IL-7 for 15 min. The phosphorylation of STAT5 was analyzed by flow cytometry. (Left) Representative data from a single experiment. Solid, unstimulated; dotted, IL-7 stimulated. (Right) Mean ± SEMs from 3 independent experiments. MFI, mean fluorescence intensity. (E) Purified B-cell subpopulations were stimulated with IL-7 for 24 hours, and expression of STAT5 target genes were determined by qRT-PCR. Results represent mean ± SEM of 3 independent experiments. (F) BM cells were pretreated with the JAK3 inhibitor (WHI-P131, 15 μM) or dimethylsulfoxide (DMSO) for 12 hours, followed by administration of COX-1 inhibitor (SC-560, 20 μM). Cells were cultured under conditions to induce B-cell differentiation for 6 days. The percentages of pre-B cells (IgM−CD43−B220+) were analyzed by flow cytometry. Numbers indicate the frequencies of total living cells. (G-H) BM cells from COX-1−/− and WT mice were transduced with lentiviral plasmid (with GFP tag) expressing (G) constitutively activated STAT5 or (H) Pax5, and empty vector CPPT was used as the control. Infected cells were cultured under conditions for 6 days to induce B-cell differentiation. The percentages of total B cells and pre-B cells among GFP+ cells were evaluated by flow cytometric analysis. (F-H) (Left) Representative from a single experiment. (Right) Mean ± SEMs from 3 independent experiments. *P < .05 and **P < .01 using unpaired Student t tests and 1-way ANOVA.

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