Figure 1.
asrij KO mice develop a myeloproliferative disorder. (A) Flow cytometric detection of Asrij expression in LT-HSCs (LSK CD150+CD48−). Percentage of LT-HSCs positive for Asrij is detailed in the histogram overlay. Mean fluorescent intensity values for “FMO control” = 232 and “Asrij stained” = 506 (n = 3 wild-type mice). (B) HSCs (LSK CD150+) were immunostained with antibodies against Asrij (green), various endosome (Rab4, Rab5, Rab11), and mitochondrial (CoxIV) markers (red) and DAPI to mark nuclei (blue). Insets show magnified view of the boxed region. Graphs indicate extent of colocalization in a single confocal plane. Bar represents 2 µm (n = 30 cells from 3 wild-type mice). (C) Schematic showing strategy for generation of floxed allele of asrij at exon 6. Black rectangles, exons; blue triangles, loxP sites; light blue triangles, frt sites; lime green rectangles, neomycin cassette; 5′ probe and 3′ probe, probes for Southern blotting; black arrows, genotyping primers (Arj_F1, Arj_F2, and Arj_R1). (D-P) Floxed and KO cells were analyzed in all cases and are indicated in red and light green, respectively. (D) Graphs show change in PB cell counts (red blood cells, WBCs, and platelets) of floxed (red circles) and KO (green triangles) mice from 2 to 12 months of age. Statistically significant differences determined using repeated measures ANOVA are indicated (n = 10 mice per genotype). (E) Representative flow cytometry plots to show the frequency of Lin− cells, LKs, and LSKs at 6 months of age (n = 7 mice per genotype). (F) Graphs showing frequency of LKs and LSKs within BM and their absolute number per femur and tibia (n = 7 mice per genotype). (G) Flow cytometric analysis of the frequencies of LT-HSCs, ST-HSCs, and MPPs at 6 months of age, identified using SLAM markers CD150/CD48 (n = 7 mice per genotype). (H-I) Graphs showing frequency of LT-HSCs, ST-HSCs, MPP2, and MPP3 within LSK and their absolute number per femur and tibia (n = 7 mice per genotype). (J) Flow cytometric analysis of CMPs, GMPs, and MEPs at 6 months of age (n = 3 mice per genotype). (K-L) Graphs showing frequency of CMPs, GMPs, MEPs and common lymphoid progenitors within BM and their absolute number per femur and tibia (n = 3 mice per genotype). (M) Graph representing frequency of BM CD11b+ and CD19+ subpopulations at 6 months of age (n = 3 mice per genotype). (F,H,I,K,L,M) Statistically significant differences for determined using ANOVA: single factor analysis are indicated. (N) Hematoxylin and eosin staining of BM sections from 2-month-old mice. Right panels show magnified view of the boxed area. Arrow points to osteolytic lesions in KO bone (n = 3 mice per genotype). Bar represents 1 mm. (O) Graph showing change in spleen weight across different age groups. Representative spleen images are shown above the respective age groups. Bar represents 0.5 cm or 1 cm, as indicated (n > 7 mice per genotype). Bars denote standard error of mean. *P < .05, **P < .01, ***P < .001. LK, LIN−c-Kit+Sca-1−; LSK, LIN−c-Kit+Sca-1+.

asrij KO mice develop a myeloproliferative disorder. (A) Flow cytometric detection of Asrij expression in LT-HSCs (LSK CD150+CD48−). Percentage of LT-HSCs positive for Asrij is detailed in the histogram overlay. Mean fluorescent intensity values for “FMO control” = 232 and “Asrij stained” = 506 (n = 3 wild-type mice). (B) HSCs (LSK CD150+) were immunostained with antibodies against Asrij (green), various endosome (Rab4, Rab5, Rab11), and mitochondrial (CoxIV) markers (red) and DAPI to mark nuclei (blue). Insets show magnified view of the boxed region. Graphs indicate extent of colocalization in a single confocal plane. Bar represents 2 µm (n = 30 cells from 3 wild-type mice). (C) Schematic showing strategy for generation of floxed allele of asrij at exon 6. Black rectangles, exons; blue triangles, loxP sites; light blue triangles, frt sites; lime green rectangles, neomycin cassette; 5′ probe and 3′ probe, probes for Southern blotting; black arrows, genotyping primers (Arj_F1, Arj_F2, and Arj_R1). (D-P) Floxed and KO cells were analyzed in all cases and are indicated in red and light green, respectively. (D) Graphs show change in PB cell counts (red blood cells, WBCs, and platelets) of floxed (red circles) and KO (green triangles) mice from 2 to 12 months of age. Statistically significant differences determined using repeated measures ANOVA are indicated (n = 10 mice per genotype). (E) Representative flow cytometry plots to show the frequency of Lin− cells, LKs, and LSKs at 6 months of age (n = 7 mice per genotype). (F) Graphs showing frequency of LKs and LSKs within BM and their absolute number per femur and tibia (n = 7 mice per genotype). (G) Flow cytometric analysis of the frequencies of LT-HSCs, ST-HSCs, and MPPs at 6 months of age, identified using SLAM markers CD150/CD48 (n = 7 mice per genotype). (H-I) Graphs showing frequency of LT-HSCs, ST-HSCs, MPP2, and MPP3 within LSK and their absolute number per femur and tibia (n = 7 mice per genotype). (J) Flow cytometric analysis of CMPs, GMPs, and MEPs at 6 months of age (n = 3 mice per genotype). (K-L) Graphs showing frequency of CMPs, GMPs, MEPs and common lymphoid progenitors within BM and their absolute number per femur and tibia (n = 3 mice per genotype). (M) Graph representing frequency of BM CD11b+ and CD19+ subpopulations at 6 months of age (n = 3 mice per genotype). (F,H,I,K,L,M) Statistically significant differences for determined using ANOVA: single factor analysis are indicated. (N) Hematoxylin and eosin staining of BM sections from 2-month-old mice. Right panels show magnified view of the boxed area. Arrow points to osteolytic lesions in KO bone (n = 3 mice per genotype). Bar represents 1 mm. (O) Graph showing change in spleen weight across different age groups. Representative spleen images are shown above the respective age groups. Bar represents 0.5 cm or 1 cm, as indicated (n > 7 mice per genotype). Bars denote standard error of mean. *P < .05, **P < .01, ***P < .001. LK, LIN−c-Kit+Sca-1−; LSK, LIN−c-Kit+Sca-1+.

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