![Hematopoietic phenotype of Calr-deficient mice. (A) Average complete blood cell counts (n = 30 in each group). (B) Hematopoietic compartment of PB assessed with flow cytometry. At 16 weeks of age, the proportion of myeloid cells (Mac1+ or Gr1+), B cells (B220+), and T cells (CD3+) was comparable among the 3 groups. (C) Histology of BM. Mouse BM was stained with hematoxylin and eosin at 6 months of age. (D) Macroscopic findings of a femur and a BM cell pellet taken from a mouse of each genotype. (E) Proportions of myeloid cells (Mac1 or Gr1), T cells (CD3+), B cells (B220+), erythroid cells (CD71+ and Ter119+), and megakaryocytes (CD41+) in BM. In BM cells from Mx1-cre;Calrf/− mice (n = 13), erythroid cells decreased significantly to less than half compared with BM cells from Mx1-cre;Calr+/+ mice (n = 8) or Mx1-cre;Calr+/− mice (n = 9) (top). No significant difference was found in the other fractions. Proportions of HSCs and progenitors in BM (bottom). Compared with Mx1-cre;Calr+/+ mice, Mx1-cre;Calrf/− mice showed increased frequencies of MPPs and GMPs. No differences were found in the frequencies of any progenitors (LT-HSCs, short-term-HSCs, LSK cells, common myeloid progenitors, erythromegakaryocyte progenitors ([MEPs]MEP), and megakaryocyte progenitors [MKPs]), between Mx1-cre;Calr+/− mice and Mx1-cre;Calr+/+ mice. (F) Number of hematopoietic colonies from BM cells. BM cells (2 × 104) were used for burst-forming unit-erythroids (BFU-Es), CFU-GM, and CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU-GEMM) colonies. BM (1 × 105) cells were used for CFU-E colonies. The number of CFU-GM was significantly higher in Mx1-cre;Calrf/− mice (n = 12) than in Mx1-cre;Calr+/+ mice (n = 9). The number of CFU-E was significantly lower in Mx1-cre;Calrf/− mice than in Mx1-cre;Calr+/+ mice and Mx1-cre;Calr+/− mice (n = 10). All data are presented as the means ± SEM. To assess statistical significance among groups, 1-way ANOVA followed by the Tukey-Kramer test was used. (G) MPO mRNA and protein expression. (left) Real-time PCR analysis of Mpo (left; n = 3 in each group). Mac1+/Gr1+ bone marrow (BM) cells were analyzed for levels of MPO expression with flow cytometry (right). One representative experiment of 3 is shown. *P < .05; **P < .01; n.s., not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/136/1/10.1182_blood.2019003358/4/bloodbld2019003358f1.png?Expires=1735839676&Signature=N84IP8bX04~Ep1gStO8lF8K8o10j2PExC2P9diGDnw0~zfyoPMFUkZQ82V3a8k6Z~2a7Mx5o6tZmm34uxhGShDh3rWnrxFNiUlAthGHJaQ-ZyU9TIrK4PDdU09RDvRRyuoQptdNUHazhWSdLTF-~5Zr-CyhKnns~FLk2r6PbUFqhBJuMyUTA~6elZPhgi4JNm-fOuTQeKEU4enB6MGgR-XAPECXWEIN6Y5es9qxaQCA26IXmQA0mpVwFF8GodDBT1C7s7-zBlTZDja1Nvfe6ZWn9UOd6d9sC0zVyCtD3tToVucWkVWkyiYQ7cxtggSjcF-Lg0W4HrVFEy3POpvpmOQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Hematopoietic phenotype of Calr-deficient mice. (A) Average complete blood cell counts (n = 30 in each group). (B) Hematopoietic compartment of PB assessed with flow cytometry. At 16 weeks of age, the proportion of myeloid cells (Mac1+ or Gr1+), B cells (B220+), and T cells (CD3+) was comparable among the 3 groups. (C) Histology of BM. Mouse BM was stained with hematoxylin and eosin at 6 months of age. (D) Macroscopic findings of a femur and a BM cell pellet taken from a mouse of each genotype. (E) Proportions of myeloid cells (Mac1 or Gr1), T cells (CD3+), B cells (B220+), erythroid cells (CD71+ and Ter119+), and megakaryocytes (CD41+) in BM. In BM cells from Mx1-cre;Calrf/− mice (n = 13), erythroid cells decreased significantly to less than half compared with BM cells from Mx1-cre;Calr+/+ mice (n = 8) or Mx1-cre;Calr+/− mice (n = 9) (top). No significant difference was found in the other fractions. Proportions of HSCs and progenitors in BM (bottom). Compared with Mx1-cre;Calr+/+ mice, Mx1-cre;Calrf/− mice showed increased frequencies of MPPs and GMPs. No differences were found in the frequencies of any progenitors (LT-HSCs, short-term-HSCs, LSK cells, common myeloid progenitors, erythromegakaryocyte progenitors ([MEPs]MEP), and megakaryocyte progenitors [MKPs]), between Mx1-cre;Calr+/− mice and Mx1-cre;Calr+/+ mice. (F) Number of hematopoietic colonies from BM cells. BM cells (2 × 104) were used for burst-forming unit-erythroids (BFU-Es), CFU-GM, and CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU-GEMM) colonies. BM (1 × 105) cells were used for CFU-E colonies. The number of CFU-GM was significantly higher in Mx1-cre;Calrf/− mice (n = 12) than in Mx1-cre;Calr+/+ mice (n = 9). The number of CFU-E was significantly lower in Mx1-cre;Calrf/− mice than in Mx1-cre;Calr+/+ mice and Mx1-cre;Calr+/− mice (n = 10). All data are presented as the means ± SEM. To assess statistical significance among groups, 1-way ANOVA followed by the Tukey-Kramer test was used. (G) MPO mRNA and protein expression. (left) Real-time PCR analysis of Mpo (left; n = 3 in each group). Mac1+/Gr1+ bone marrow (BM) cells were analyzed for levels of MPO expression with flow cytometry (right). One representative experiment of 3 is shown. *P < .05; **P < .01; n.s., not significant.