American Society of Hematology

Figure 6

$Figure 6. JAK2V617F+ cells escape from lipocalin-2–induced growth suppression resulting from attenuated p53 activation. (A) Intracellular levels of ROS in 32D cells transduced with Kusabira-Orange empty vector or JAK2V617F-IRES-Kusabira-Orange were measured by H2DCFDA dye assay following exposure to 100 ng/mL recombinant lipocalin-2 for 24 hours. Average mean FITC fluorescence intensity is shown (n = 4 each). (B) Relative numbers of 32D cells with empty vector or JAK2V617F after 36 hours incubation with or without 100 ng/mL lipocalin-2 (n = 4 each). (C) Average apoptotic cell ratio in JAK2V617F-32D cells treated with each CM (n = 4 each). (D) Colony-forming cell assay for Lcn2-deficient murine bone marrow c-Kit+ cells transduced with empty vector or JAK2V617F with or without 100 ng/mL lipocalin-2 (n = 4 each). (E) Relative cell numbers of normal bone marrow c-Kit+ cells 48 hours after incubation with wild-type or Lcn2−/− bone marrow cells transduced with JAK2V617F-IRES-GFP (n = 4 each). In coculture experiments, the number of normal bone marrow cells was counted by multiplying the total number of cells by the fraction of GFP– cells. (F) Immunoblotting of p53 and phospho-p53 in Lcn2-deficient murine bone marrow c-Kit+ cells transduced with empty vector or JAK2V617F with or without 100 ng/mL lipocalin-2. (G) Colony-forming cell assay for bone marrow CD34+ cells from patients with MPN and non-MPN control patients with or without 1 μg/mL recombinant lipocalin-2. Cells were seeded in semisolid medium (Methocult H4434 Classic; StemCell Technologies). The total numbers of colonies per 1000 cells are shown. The colony numbers were compared between wells with or without lipocalin-2 by using paired Student t tests. Detailed patient characteristics are described in supplemental Table 3. (H) Immunoblotting of MDM2 in 32D cells with empty vector or JAK2V617F. (I) Relative cell numbers of 32D cells with empty vector or JAK2V617F after 36 hours incubation with or without 100 ng/mL lipocalin-2 and 0.5 μM nutlin-3 (n = 4 each). Error bars indicate SD.$

JAK2V617F⁺ cells escape from lipocalin-2–induced growth suppression resulting from attenuated p53 activation. (A) Intracellular levels of ROS in 32D cells transduced with Kusabira-Orange empty vector or JAK2V617F-IRES-Kusabira-Orange were measured by H2DCFDA dye assay following exposure to 100 ng/mL recombinant lipocalin-2 for 24 hours. Average mean FITC fluorescence intensity is shown (n = 4 each). (B) Relative numbers of 32D cells with empty vector or JAK2V617F after 36 hours incubation with or without 100 ng/mL lipocalin-2 (n = 4 each). (C) Average apoptotic cell ratio in JAK2V617F-32D cells treated with each CM (n = 4 each). (D) Colony-forming cell assay for Lcn2-deficient murine bone marrow c-Kit⁺ cells transduced with empty vector or JAK2V617F with or without 100 ng/mL lipocalin-2 (n = 4 each). (E) Relative cell numbers of normal bone marrow c-Kit⁺ cells 48 hours after incubation with wild-type or Lcn2^−/− bone marrow cells transduced with JAK2V617F-IRES-GFP (n = 4 each). In coculture experiments, the number of normal bone marrow cells was counted by multiplying the total number of cells by the fraction of GFP^– cells. (F) Immunoblotting of p53 and phospho-p53 in Lcn2-deficient murine bone marrow c-Kit⁺ cells transduced with empty vector or JAK2V617F with or without 100 ng/mL lipocalin-2. (G) Colony-forming cell assay for bone marrow CD34⁺ cells from patients with MPN and non-MPN control patients with or without 1 μg/mL recombinant lipocalin-2. Cells were seeded in semisolid medium (Methocult H4434 Classic; StemCell Technologies). The total numbers of colonies per 1000 cells are shown. The colony numbers were compared between wells with or without lipocalin-2 by using paired Student t tests. Detailed patient characteristics are described in supplemental Table 3. (H) Immunoblotting of MDM2 in 32D cells with empty vector or JAK2V617F. (I) Relative cell numbers of 32D cells with empty vector or JAK2V617F after 36 hours incubation with or without 100 ng/mL lipocalin-2 and 0.5 μM nutlin-3 (n = 4 each). Error bars indicate SD.

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