Figure 3.
Murine DPT is induced by conditioning, and supranormal levels of DPT affect the homing and engraftment. (A) qRT-PCR of Dpt expression in marrow (11-Gy radiation; n = 6 animals per group) and spleen (9-Gy radiation; n = 5 animals per group). (B) ELISA of plasma DPT expression in mice after 9-Gy radiation (n = 4-6 animals per time point). (C) Western blot of murine plasma DPT after 11-Gy radiation, with band semiquantification shown below. (D) Western blot of murine plasma DPT after treatment with LPS to induce inflammation, with band semiquantification shown below. (E) Schema for (F-I). CD45.2 animals were injected IV via the lateral tail vein with 2 μg of murine rDPT 24 hours after receiving 9-Gy radiation. Following rDPT delivery, 2 million EGFP+ WBMs were injected in the contralateral tail vein. (F) Western blot of plasma DPT (native) and rDPT following IV injection of rDPT. The rDPT is slightly smaller than native DPT, as shown by a shift in molecular weight. (G) Number of donor cells homed to the marrow, as determined by flow cytometry 16 hours after transplant (n = 5-8 animals per group). Gating strategy is shown in supplemental Figure 3. (H) Peripheral blood (PB) CFU-C assessed 16 hours after transplant (n = 6 animals per group). (I) CFU-spleen assessed 8 days after transplant. Photographs show examples of harvested spleens after fixation with Bouin’s solution (left and middle panels). Scale bars, 5 mm. Each dot represents an individual animal (right panel). (J) The number of donor hematopoietic CFU-C in the marrow was assessed 8 days after transplant. (K) Schema for primary and secondary HCT. CD45.2 mice were injected IV via the lateral tail vein with 2 μg of murine rDPT 24 hours after sublethal (4-Gy) radiation. Within 30 minutes following rDPT delivery, 2 million CD45.1 donor WBM cells were injected in the contralateral tail vein. (L) Peripheral CD45.1 donor engraftment after the primary transplant (n = 10-12 animals per group). (M) Flow cytometry gating strategy for evaluation of donor LSK engraftment at 4 months after primary HCT. (N) Peripheral donor engraftment after secondary transplant (n = 9-10 animals per group). All data are shown as means and standard deviation, unless otherwise noted. *P < .05, **P < .01, Student t test. BMMC, bone marrow mononuclear cell; BSA, bovine serum albumin; FSC, forward scatter; L, left; PBMC, peripheral blood mononuclear cell; PI, propidium iodide; Pre, pretreatment; R, right; SSC, side scatter.

Murine DPT is induced by conditioning, and supranormal levels of DPT affect the homing and engraftment. (A) qRT-PCR of Dpt expression in marrow (11-Gy radiation; n = 6 animals per group) and spleen (9-Gy radiation; n = 5 animals per group). (B) ELISA of plasma DPT expression in mice after 9-Gy radiation (n = 4-6 animals per time point). (C) Western blot of murine plasma DPT after 11-Gy radiation, with band semiquantification shown below. (D) Western blot of murine plasma DPT after treatment with LPS to induce inflammation, with band semiquantification shown below. (E) Schema for (F-I). CD45.2 animals were injected IV via the lateral tail vein with 2 μg of murine rDPT 24 hours after receiving 9-Gy radiation. Following rDPT delivery, 2 million EGFP+ WBMs were injected in the contralateral tail vein. (F) Western blot of plasma DPT (native) and rDPT following IV injection of rDPT. The rDPT is slightly smaller than native DPT, as shown by a shift in molecular weight. (G) Number of donor cells homed to the marrow, as determined by flow cytometry 16 hours after transplant (n = 5-8 animals per group). Gating strategy is shown in supplemental Figure 3. (H) Peripheral blood (PB) CFU-C assessed 16 hours after transplant (n = 6 animals per group). (I) CFU-spleen assessed 8 days after transplant. Photographs show examples of harvested spleens after fixation with Bouin’s solution (left and middle panels). Scale bars, 5 mm. Each dot represents an individual animal (right panel). (J) The number of donor hematopoietic CFU-C in the marrow was assessed 8 days after transplant. (K) Schema for primary and secondary HCT. CD45.2 mice were injected IV via the lateral tail vein with 2 μg of murine rDPT 24 hours after sublethal (4-Gy) radiation. Within 30 minutes following rDPT delivery, 2 million CD45.1 donor WBM cells were injected in the contralateral tail vein. (L) Peripheral CD45.1 donor engraftment after the primary transplant (n = 10-12 animals per group). (M) Flow cytometry gating strategy for evaluation of donor LSK engraftment at 4 months after primary HCT. (N) Peripheral donor engraftment after secondary transplant (n = 9-10 animals per group). All data are shown as means and standard deviation, unless otherwise noted. *P < .05, **P < .01, Student t test. BMMC, bone marrow mononuclear cell; BSA, bovine serum albumin; FSC, forward scatter; L, left; PBMC, peripheral blood mononuclear cell; PI, propidium iodide; Pre, pretreatment; R, right; SSC, side scatter.

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