Figure 2.
Figure 2. Distinct RTKs determine growth and survival of ALL. (A) Tyrosine phosphorylation of RTKs in primary ALLs, XT-ALLs, and ALL cell lines before and after ligand stimulation determined by quantitative sandwich ELISA. Mean values (plus or minus range) of technical duplicates are shown. Experiments were reproduced independently using biological replicates with similar results except for primary ALL due to limited material. (B) MTT assay of primary ALL cells after stimulation with RTK-specific ligands. Relative proliferation over time (days) normalized to nonstimulated controls. Combination: Simultaneous application of ligands FLT3L, platelet-derived growth factor β chain homodimer (PDGF-BB), and IGF-1 according to surface expression of corresponding RTK in individual ALL. Assays were performed in triplicate. Mean values plus or minus SD are shown. (C) Heat map of RTK expression in a panel of established XT-ALLs determined by ELISA. Color scale bar indicates absolute amount of protein (femtograms) per microgram of protein lysate. (D) Proliferation of selected XT-ALL under FLT3 or PDGF-BB stimulation over time normalized to nonstimulated control using MTT assay. Mean values plus or minus SD are shown. (E) In vivo growth and survival of PDX-ALLs (XT-ALL22089, XT-ALL109, XT-ALL114) upon FLT3 (sh4FLT3) or PDGFRB (sh2PDGF)-directed RNAi compared with scrambled shRNA (sh1SCR). Dot plots represent mean percentage plus or minus SD of YFP-reporter positive XT-ALL cells in bone marrow of euthanized animals at the onset of overt clinical disease 6 to 8 weeks after transplantation. Competitive leukemia cell engraftment and progression of disease were monitored by YFP reporter fluorescence of shRNA-expressing target cells in peripheral blood in relation to the total human CD45+ leukemia population (supplemental Figure 4E-F). Number of mice per cohort is indicated. Mean values plus or minus SD are shown (2-tailed Mann-Whitney U test; **P < .01; ***P < .001). BCP-ALL, B-cell precursor ALL; SCF, stem cell factor; VEGF, vascular endothelial growth factor.

Distinct RTKs determine growth and survival of ALL. (A) Tyrosine phosphorylation of RTKs in primary ALLs, XT-ALLs, and ALL cell lines before and after ligand stimulation determined by quantitative sandwich ELISA. Mean values (plus or minus range) of technical duplicates are shown. Experiments were reproduced independently using biological replicates with similar results except for primary ALL due to limited material. (B) MTT assay of primary ALL cells after stimulation with RTK-specific ligands. Relative proliferation over time (days) normalized to nonstimulated controls. Combination: Simultaneous application of ligands FLT3L, platelet-derived growth factor β chain homodimer (PDGF-BB), and IGF-1 according to surface expression of corresponding RTK in individual ALL. Assays were performed in triplicate. Mean values plus or minus SD are shown. (C) Heat map of RTK expression in a panel of established XT-ALLs determined by ELISA. Color scale bar indicates absolute amount of protein (femtograms) per microgram of protein lysate. (D) Proliferation of selected XT-ALL under FLT3 or PDGF-BB stimulation over time normalized to nonstimulated control using MTT assay. Mean values plus or minus SD are shown. (E) In vivo growth and survival of PDX-ALLs (XT-ALL22089, XT-ALL109, XT-ALL114) upon FLT3 (sh4FLT3) or PDGFRB (sh2PDGF)-directed RNAi compared with scrambled shRNA (sh1SCR). Dot plots represent mean percentage plus or minus SD of YFP-reporter positive XT-ALL cells in bone marrow of euthanized animals at the onset of overt clinical disease 6 to 8 weeks after transplantation. Competitive leukemia cell engraftment and progression of disease were monitored by YFP reporter fluorescence of shRNA-expressing target cells in peripheral blood in relation to the total human CD45+ leukemia population (supplemental Figure 4E-F). Number of mice per cohort is indicated. Mean values plus or minus SD are shown (2-tailed Mann-Whitney U test; **P < .01; ***P < .001). BCP-ALL, B-cell precursor ALL; SCF, stem cell factor; VEGF, vascular endothelial growth factor.

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