Figure 7
Figure 7. Knockdown p38 MAPK-STAT1 by genetic modification significantly reversed the enhanced neutrophil development caused by Wip1 deficiency. (A) Erythrocyte-depleted BMCs of WT and p38+/− mice were treated with G-CSF, and the expression of p38 MAPK, STAT1, and C/EBPα was detected by immunoblotting. (B) A total of 500 GMPs sorted from WT and Wip1KO mice were seeded in methylcellulose media supplemented with SCF, IL-3, and G-CSF. The experiment was performed in triplicate. One representative CFU generated from WT or p38+/− GMPs was photographed. Absolute neutrophil number per CFU (C), number of CFUs (D), and neutrophil number of per 500 GMPs (E) were detected after 8 days in culture. Data (mean ± SD) are one of 2 independent experiments with similar results. *P < .05, between the indicated groups. **P < .01, between the indicated groups.***P < .001, between the indicated groups. (F) Sorted GMPs of WT and Wip1KO mice were transfected with control and STAT1 shRNA vectors and further treated with G-CSF. The expression of STAT1 and C/EBPα was detected by Western blotting. (G-J) A total of 500 sorted WT and Wip1-deficient GMPs expressing either control or STAT1 shRNA vector were seeded in methylcellulose media supplemented with SCF, IL-3, and G-CSF; the colony-forming assays were then performed. One representative CFU generated in each group was photographed (G). Absolute neutrophil number per CFU (H), number of CFUs (I), and neutrophil number of per 500 GMPs (J) were detected after 8 days in culture. Data (mean ± SD) are one of 2 independent experiments with similar results. *P < .05, between the indicated groups. **P < .01, between the indicated groups. ***P < .001, between the indicated groups.

Knockdown p38 MAPK-STAT1 by genetic modification significantly reversed the enhanced neutrophil development caused by Wip1 deficiency. (A) Erythrocyte-depleted BMCs of WT and p38+/− mice were treated with G-CSF, and the expression of p38 MAPK, STAT1, and C/EBPα was detected by immunoblotting. (B) A total of 500 GMPs sorted from WT and Wip1KO mice were seeded in methylcellulose media supplemented with SCF, IL-3, and G-CSF. The experiment was performed in triplicate. One representative CFU generated from WT or p38+/− GMPs was photographed. Absolute neutrophil number per CFU (C), number of CFUs (D), and neutrophil number of per 500 GMPs (E) were detected after 8 days in culture. Data (mean ± SD) are one of 2 independent experiments with similar results. *P < .05, between the indicated groups. **P < .01, between the indicated groups.***P < .001, between the indicated groups. (F) Sorted GMPs of WT and Wip1KO mice were transfected with control and STAT1 shRNA vectors and further treated with G-CSF. The expression of STAT1 and C/EBPα was detected by Western blotting. (G-J) A total of 500 sorted WT and Wip1-deficient GMPs expressing either control or STAT1 shRNA vector were seeded in methylcellulose media supplemented with SCF, IL-3, and G-CSF; the colony-forming assays were then performed. One representative CFU generated in each group was photographed (G). Absolute neutrophil number per CFU (H), number of CFUs (I), and neutrophil number of per 500 GMPs (J) were detected after 8 days in culture. Data (mean ± SD) are one of 2 independent experiments with similar results. *P < .05, between the indicated groups. **P < .01, between the indicated groups. ***P < .001, between the indicated groups.

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