American Society of Hematology

Figure 4

$Figure 4. The shortest isoform of LSD1 enhances the stemness and self-renewal of HSPCs in vivo. (A) Top panel, schematic view of the injected fragment for the generation of LSD1 transgenic mice. Bottom panel, we quantified the expression of human LSD1 and murine GAPDH (internal control) transcripts in 2 lines of LSD1 transgenic and B6J (Control) mice using specific primers (supplemental Table 1). Right panel, genomic DNA (5 µg) was digested with BamHI and hybridized with LSD1 cDNA as a probe to estimate copy numbers of transgene. Lane 1, DNA from wild-type mice + 2 copies of the injection fragment; lane 2, DNA from wild-type mice + 20 copies of the injection fragment; lane 3, DNA from wild-type mice; lane 4, DNA from line 6 mice; lane 5, DNA from line 3 mice. (B) We determined the percentage of LSK and Lin−/Sca-1+ cells in the bone marrow of 2 lines of LSD1 transgenic and B6J (Control) mice using flow cytometry. Representative data are shown in panel ii (P8 and P6 correspond to LSK and Lin−/Sca-1+ fractions, respectively). (C) We determined the percentage of CD150+/CD48− cells in LSK fractions of 2 lines of LSD1 transgenic and B6J mice using flow cytometry. Representative data are shown in panel ii (the boxed area corresponds to CD150+/CD48−/LSK fractions). (D) We determined the cell cycle profile of Lin−/Sca-1+ and Lin− bone marrow mononuclear cells in 2 lines of LSD1 transgenic and B6J (Control) mice in vivo. (E) Bone marrow cells from 2 lines of LSD1 transgenic and B6J (Control) mice (CD45.2) were transplanted into lethally irradiated C57BL/6 (CD45.1) mice. We determined the percentage of CD45.2+ cells in the peripheral blood 24 weeks after transplantation. (F) Bone marrow cells were collected from control- and LSD1-recipient mice 12 weeks after primary transplantation, and transplanted into lethally irradiated C57BL/6 mice. Tertiary transplantation was similarly performed 12 weeks after secondary transplantation. The means ± SD (bars) of 3 independent experiments are shown. *P < .05 and **P < .01 determined by 1-way ANOVA with the Bonferroni post hoc test. (G) Cell cycle status of HSPCs in LSD1 transgenic mice (see “Results” for details).$

The shortest isoform of LSD1 enhances the stemness and self-renewal of HSPCs in vivo. (A) Top panel, schematic view of the injected fragment for the generation of LSD1 transgenic mice. Bottom panel, we quantified the expression of human LSD1 and murine GAPDH (internal control) transcripts in 2 lines of LSD1 transgenic and B6J (Control) mice using specific primers (supplemental Table 1). Right panel, genomic DNA (5 µg) was digested with BamHI and hybridized with LSD1 cDNA as a probe to estimate copy numbers of transgene. Lane 1, DNA from wild-type mice + 2 copies of the injection fragment; lane 2, DNA from wild-type mice + 20 copies of the injection fragment; lane 3, DNA from wild-type mice; lane 4, DNA from line 6 mice; lane 5, DNA from line 3 mice. (B) We determined the percentage of LSK and Lin⁻/Sca-1⁺ cells in the bone marrow of 2 lines of LSD1 transgenic and B6J (Control) mice using flow cytometry. Representative data are shown in panel ii (P8 and P6 correspond to LSK and Lin⁻/Sca-1⁺ fractions, respectively). (C) We determined the percentage of CD150⁺/CD48⁻ cells in LSK fractions of 2 lines of LSD1 transgenic and B6J mice using flow cytometry. Representative data are shown in panel ii (the boxed area corresponds to CD150⁺/CD48⁻/LSK fractions). (D) We determined the cell cycle profile of Lin⁻/Sca-1⁺ and Lin⁻ bone marrow mononuclear cells in 2 lines of LSD1 transgenic and B6J (Control) mice in vivo. (E) Bone marrow cells from 2 lines of LSD1 transgenic and B6J (Control) mice (CD45.2) were transplanted into lethally irradiated C57BL/6 (CD45.1) mice. We determined the percentage of CD45.2⁺ cells in the peripheral blood 24 weeks after transplantation. (F) Bone marrow cells were collected from control- and LSD1-recipient mice 12 weeks after primary transplantation, and transplanted into lethally irradiated C57BL/6 mice. Tertiary transplantation was similarly performed 12 weeks after secondary transplantation. The means ± SD (bars) of 3 independent experiments are shown. *P < .05 and **P < .01 determined by 1-way ANOVA with the Bonferroni post hoc test. (G) Cell cycle status of HSPCs in LSD1 transgenic mice (see “Results” for details).

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