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. Author manuscript; available in PMC: 2017 Apr 1.
Published in final edited form as: Stem Cells. 2016 Feb 2;34(4):1068–1082. doi: 10.1002/stem.2293

Figure 1. Flcn deletion in bone marrow disrupts normal hematopoiesis.

Figure 1

(A) Bone marrow cells (BMC) and splenocytes (SPC) were isolated from Flcnf/f;Mx1-Cre− (Flcn WT) and Flcnf/f;Mx1-Cre+(Flcn KO) mice on day 5 and day 21 after pIpC treatment. PCR genotyping demonstrated efficient deletion of Flcn in bone marrow (BM) and spleen (SP). (B) Flcn mRNA expression in BM was quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on day 5 and day 21 after pIpC treatment (Y-axis). BM day 5, BM day 21 and SP day 21: n=5 mice for each genotype; SP day 5:n=3 mice for each genotype. Data are presented as mean with SD (unpaired student’s t-test: ****P<.0001, ***P<.001). (C) Efficient suppression of Flcn protein expression was also confirmed by western blotting on day 5 and day 21 after pIpC treatment. (D) Kaplan-Meier survival analysis shows a statistically significant difference between control and Flcn KO (log-rank test: ****P<.0001). Median survival time of Flcn KO mice is 27 days after pIpC treatment (n=9). Dotted lines indicate SEM. (E) Gross images of spleens from Flcn WT and Flcn KO mice at day 28 after pIpC treatment show symmetrical enlargement of Flcn KO spleen compared with Flcn WT spleen. One representative image of more than 10 mice is shown. (F) % spleen/body weight (BW); spleen weight/BW × 100 was calculated at different day after pIpC treatment. The mean % spleen/body weights of Flcn WT and Flcn KO mice are 0.64±0.18% vs 1.47±0.22% (**P =.01) at day 7 (1W), 0.33±0.08% vs 3.68±0.89% (***P =.0003) at day 14 (2W) and 0.37±0.09% vs 8.34±2.27% (****P <.0001) at day 21 (3W) determined using unpaired student’s t-test. n=4 for each group on day 7 and 14, n=5 for each group on day 21. Data are represented as means ± SD. (G) Gross images of femurs and tibiae from Flcn WT and Flcn KO mice at day 21 after pIpC treatment show pale BM in Flcn KO mice. One representative image of more than 10 mice for each genotype is shown. (H) H & E staining of bone marrow from Flcn WT and Flcn KO mice at day 21 after pIpC treatment shows hypoplastic Flcn KO BM. One representative image of 3 mice for each genotype. (I) Absolute cell numbers of BMC from femurs and tibiae of Flcn WT and Flcn KO mice at day 21 (5.48±1.27 × 107 vs 0.35±0.06 × 107, unpaired student’s t-test: ****P <.0001). n=5 for each group. Data are represented as means± SD.