Figure 1.
CD53 expression rapidly increases in HSCs during stress. (A) Relative microarray expression values of Cd53 from sorted BM and spleen HSCs (KSL SLAM) from WT mice treated with the TLR2 agonist PAM3CSK4 (red; 25 μg injected intraperitoneally every 48 hours for 3 doses and analyzed 24 hours after the final dose), the CXCR4 antagonist Pol5551 (blue; 100 mg/kg per day for 2 weeks, delivered by osmotic pump), or the recombinant cytokine G-CSF (green; 3.75 μg injected subcutaneously every 12 hours for 7 days and analyzed 2 hours after the final dose) compared with vehicle-treated controls (orange dashed line). (B) Cd53 expression by qRT-PCR of RNA isolated from sorted HSCs after treatment with G-CSF (green; as above) or 5-FU (blue; 150 mg/kg injected intraperitoneally 6 days before analysis), compared with vehicle-treated controls. Relative quantitation of CD53 was calculated against actin using the 2−ΔCt method. (C) Human CD53 mRNA expression in G-CSF- and BL-8040-mobilized CD34+ cells compared with that in untreated CD34+ BM cells. Relative quantitation of CD53 was calculated against actin using 2−ΔCt method. (D) Outline of kinetic G-CSF injection and analysis experiment. WT mice were treated with 3.75 μg G-CSF every 12 hours, for up to 5 days and analyzed at designated time points. (E-F) Surface expression of CD53 from BM and splenic HSCs at the indicated times during treatment compared with vehicle-treated controls (orange dashed line; n = 3-5 mice per group). (G) Surface expression of CD53 (red) and DAPI (blue) assessed by immunofluorescence and confocal microscopy of WT HSCs from vehicle-treated BM and G-CSF-treated mobilized spleen HSCs (7 days of G-CSF; n = 2 mice per group with at least 20 individual HSCs imaged per mouse), with quantification shown on the left and a representative image of a single z-plane on the right. Error bars represent mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001 by an unpaired Student t test. DAPI, 4',6-diamidino-2-phenylindole; nMFI, normalized median fluorescent intensity; PB, peripheral blood; Spl, spleen.

CD53 expression rapidly increases in HSCs during stress. (A) Relative microarray expression values of Cd53 from sorted BM and spleen HSCs (KSL SLAM) from WT mice treated with the TLR2 agonist PAM3CSK4 (red; 25 μg injected intraperitoneally every 48 hours for 3 doses and analyzed 24 hours after the final dose), the CXCR4 antagonist Pol5551 (blue; 100 mg/kg per day for 2 weeks, delivered by osmotic pump), or the recombinant cytokine G-CSF (green; 3.75 μg injected subcutaneously every 12 hours for 7 days and analyzed 2 hours after the final dose) compared with vehicle-treated controls (orange dashed line). (B) Cd53 expression by qRT-PCR of RNA isolated from sorted HSCs after treatment with G-CSF (green; as above) or 5-FU (blue; 150 mg/kg injected intraperitoneally 6 days before analysis), compared with vehicle-treated controls. Relative quantitation of CD53 was calculated against actin using the 2−ΔCt method. (C) Human CD53 mRNA expression in G-CSF- and BL-8040-mobilized CD34+ cells compared with that in untreated CD34+ BM cells. Relative quantitation of CD53 was calculated against actin using 2−ΔCt method. (D) Outline of kinetic G-CSF injection and analysis experiment. WT mice were treated with 3.75 μg G-CSF every 12 hours, for up to 5 days and analyzed at designated time points. (E-F) Surface expression of CD53 from BM and splenic HSCs at the indicated times during treatment compared with vehicle-treated controls (orange dashed line; n = 3-5 mice per group). (G) Surface expression of CD53 (red) and DAPI (blue) assessed by immunofluorescence and confocal microscopy of WT HSCs from vehicle-treated BM and G-CSF-treated mobilized spleen HSCs (7 days of G-CSF; n = 2 mice per group with at least 20 individual HSCs imaged per mouse), with quantification shown on the left and a representative image of a single z-plane on the right. Error bars represent mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001 by an unpaired Student t test. DAPI, 4',6-diamidino-2-phenylindole; nMFI, normalized median fluorescent intensity; PB, peripheral blood; Spl, spleen.

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