Figure 5
Figure 5. Targeting Mer in vivo delays leukemia onset and decreases CNS infiltration. (A) 697 cells were subjected to treatment with 800 nM UNC-569 for 48 hours. Cells were then treated with 70 µM pervanadate for 3 minutes to stabilize the phosphorylated Mer (PV) or left untreated (no PV) and then lysed. IPs and supernatants were analyzed for p-Mer and total Mer expression by western blotting as indicated. Control lanes: empty (lysis buffer only), AbC (antibody control: buffer, antibody for IP, contains no beads and lysate), BC (bead control: contains lysate, beads, contains no IP antibody). (B) Primary xenograft cells from 2 t(1;19) Merhigh patients (#1 and 3 in Table 1) used for secondary xenograft transplantation were analyzed for Mer, Axl, and Tyro3 expression by western blotting. The OCI-AML-5 cell line was used as a control for detection of positive bands. β-Actin was used as a loading control. (C-E) Patient Merhigh #3. (C) Twenty NSG mice were xenografted with primary cells of patient #3 (secondary transplantation of primary xenograft spleen cells). Mice were treated with DMSO or 10 mg/kg UNC-569 intraperitoneally once per day starting on the day of transplantation until day 42. The percentage of blasts in the peripheral blood was determined by FACS analysis at the indicated time points (Mann-Whitney U test). (D) Splenic volume in patient #3 mice as approximated by the formula longest length × highest height × broadest width in cm3 (Mann-Whitney U test). (E) Semiquantitative scoring of CNS status upon sacrification of the patient #3 mice (χ2 test). (F-H) Patient Merhigh #1. (F) Twenty NSG mice were xenografted with primary cells of patient #1 (secondary transplantation of primary xenograft spleen cells). Mice were treated with DMSO or 10 mg/kg UNC-569 intraperitoneally once per day starting on the day of transplantation until they showed leukemic symptoms. The graph shows survival curves (Kaplan-Meier log-rank test). (G) Approximated splenic volume in patient #1 mice (Mann-Whitney U test). (H) Semiquantitative scoring of CNS status upon sacrification of the patient #1 mice (1-sided Fisher exact test). (I) Twenty mice were xenografted with 697shGFP and 697shMer cells. One mouse in the 697shGFP group died due to procedural complications. Mice were sacrificed on day 20. The table shows the semiquantitative scoring of CNS status upon sacrification of the mice (1-sided Fisher exact test). PV, pervanadate.

Targeting Mer in vivo delays leukemia onset and decreases CNS infiltration. (A) 697 cells were subjected to treatment with 800 nM UNC-569 for 48 hours. Cells were then treated with 70 µM pervanadate for 3 minutes to stabilize the phosphorylated Mer (PV) or left untreated (no PV) and then lysed. IPs and supernatants were analyzed for p-Mer and total Mer expression by western blotting as indicated. Control lanes: empty (lysis buffer only), AbC (antibody control: buffer, antibody for IP, contains no beads and lysate), BC (bead control: contains lysate, beads, contains no IP antibody). (B) Primary xenograft cells from 2 t(1;19) Merhigh patients (#1 and 3 in Table 1) used for secondary xenograft transplantation were analyzed for Mer, Axl, and Tyro3 expression by western blotting. The OCI-AML-5 cell line was used as a control for detection of positive bands. β-Actin was used as a loading control. (C-E) Patient Merhigh #3. (C) Twenty NSG mice were xenografted with primary cells of patient #3 (secondary transplantation of primary xenograft spleen cells). Mice were treated with DMSO or 10 mg/kg UNC-569 intraperitoneally once per day starting on the day of transplantation until day 42. The percentage of blasts in the peripheral blood was determined by FACS analysis at the indicated time points (Mann-Whitney U test). (D) Splenic volume in patient #3 mice as approximated by the formula longest length × highest height × broadest width in cm3 (Mann-Whitney U test). (E) Semiquantitative scoring of CNS status upon sacrification of the patient #3 mice (χ2 test). (F-H) Patient Merhigh #1. (F) Twenty NSG mice were xenografted with primary cells of patient #1 (secondary transplantation of primary xenograft spleen cells). Mice were treated with DMSO or 10 mg/kg UNC-569 intraperitoneally once per day starting on the day of transplantation until they showed leukemic symptoms. The graph shows survival curves (Kaplan-Meier log-rank test). (G) Approximated splenic volume in patient #1 mice (Mann-Whitney U test). (H) Semiquantitative scoring of CNS status upon sacrification of the patient #1 mice (1-sided Fisher exact test). (I) Twenty mice were xenografted with 697shGFP and 697shMer cells. One mouse in the 697shGFP group died due to procedural complications. Mice were sacrificed on day 20. The table shows the semiquantitative scoring of CNS status upon sacrification of the mice (1-sided Fisher exact test). PV, pervanadate.

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