Figure 1
Figure 1. The expression of Wip1 in immune cells and phenotypic characterization of myeloid cell population of Wip1-deficient mice were determined. (A) Quantitative PCR analysis of Wip1 expression in the different subtypes of immune cell types (mean ± SD, n = 3). (B) Quantitative PCR analysis of Wip1 expression during neutrophil development from HSCs (mean ± SD, n = 3). (C) Bar graphs represent the total numbers of neutrophils in peripheral blood and BM of WT or Wip1KO mice. Values represent mean ± SD, n = 15 mice of each genotype. ***P < .001, WT versus Wip1KO. (D) Representative FACS analysis of peripheral blood and BM CD11b+Ly6G+ neutrophils. (E) Top: Morphologic analysis of peripheral blood neutrophils with Giemsa staining (original magnification ×100). Bottom: Quantitative assessment of nuclear segmentation scored in at least 50 neutrophils per animal. Blood smears from 5 mice of each genotype were morphologically analyzed and the extent of nuclear segmentation scored in at least 50 neutrophils per animals. **P < .01, WT versus Wip1KO. ***P < .001, WT versus Wip1KO. (F) Morphologic analysis of WT and Wip1-deficient neutrophils with transmission electron micrograph was shown (original magnification ×890). (G) The neutrophil defense response to bacterial infection was determined in vitro. Neutrophils isolated from WT or Wip1KO mice were cultured with E coli. The percentage of phagocytosis neutrophils was determined by FACS. The survival of bacteria in the culture was determined as described in “Methods.” (H) The E coli survival ratios were assayed after coculture of E coli with WT or Wip1KO neutrophils. The experiments were performed in triplicate. (I) Respiratory burst by isolated BM neutrophils as measured by oxidation of dihydrorhodamine 123 after activation with 40 ng/mL PMA. Data represent the mean fluorescent intensity of all cells (mean ± SD, n = 3, top). Bottom panel: Representative histogram showing the percentage of dihydrorhodamine-positive cells after incubation of neutrophils with 40 ng/mL PMA for 30 minutes. (J) Wip1KO CD11b+Ly6G+ neutrophils were increased in mixed chimeric mice, which were generated by transplanting CD45.2+ Wip1KO BMCs mixed with CD45.1+ WT BMCs at the ratio of 1:1 into lethally irradiated CD45.1+ mice. Representative results are shown from one of 3 independent experiments performed.

The expression of Wip1 in immune cells and phenotypic characterization of myeloid cell population of Wip1-deficient mice were determined. (A) Quantitative PCR analysis of Wip1 expression in the different subtypes of immune cell types (mean ± SD, n = 3). (B) Quantitative PCR analysis of Wip1 expression during neutrophil development from HSCs (mean ± SD, n = 3). (C) Bar graphs represent the total numbers of neutrophils in peripheral blood and BM of WT or Wip1KO mice. Values represent mean ± SD, n = 15 mice of each genotype. ***P < .001, WT versus Wip1KO. (D) Representative FACS analysis of peripheral blood and BM CD11b+Ly6G+ neutrophils. (E) Top: Morphologic analysis of peripheral blood neutrophils with Giemsa staining (original magnification ×100). Bottom: Quantitative assessment of nuclear segmentation scored in at least 50 neutrophils per animal. Blood smears from 5 mice of each genotype were morphologically analyzed and the extent of nuclear segmentation scored in at least 50 neutrophils per animals. **P < .01, WT versus Wip1KO. ***P < .001, WT versus Wip1KO. (F) Morphologic analysis of WT and Wip1-deficient neutrophils with transmission electron micrograph was shown (original magnification ×890). (G) The neutrophil defense response to bacterial infection was determined in vitro. Neutrophils isolated from WT or Wip1KO mice were cultured with E coli. The percentage of phagocytosis neutrophils was determined by FACS. The survival of bacteria in the culture was determined as described in “Methods.” (H) The E coli survival ratios were assayed after coculture of E coli with WT or Wip1KO neutrophils. The experiments were performed in triplicate. (I) Respiratory burst by isolated BM neutrophils as measured by oxidation of dihydrorhodamine 123 after activation with 40 ng/mL PMA. Data represent the mean fluorescent intensity of all cells (mean ± SD, n = 3, top). Bottom panel: Representative histogram showing the percentage of dihydrorhodamine-positive cells after incubation of neutrophils with 40 ng/mL PMA for 30 minutes. (J) Wip1KO CD11b+Ly6G+ neutrophils were increased in mixed chimeric mice, which were generated by transplanting CD45.2+ Wip1KO BMCs mixed with CD45.1+ WT BMCs at the ratio of 1:1 into lethally irradiated CD45.1+ mice. Representative results are shown from one of 3 independent experiments performed.

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