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. 2022 Oct 19;11:e79940. doi: 10.7554/eLife.79940

Figure 1. Conditioned media of human bone marrow stromal cells (hBMSCs) prevents apoptosis in FLT3-ITD acute myeloid leukemia (AML) cells upon FLT3 inhibition.

(A, B), FLT3-ITD AML cell lines, MOLM-13 and MV4-11, were treated with either DMSO (veh) or quizartinib with indicated doses for 48 hr in regular media (RPMI) or 50% conditioned media of human BM stromal cells in RPMI (hBMSC-CM). (A) Apoptotic cells were detected by 7-AAD/Annexin V staining post-treatment. (B) Cell viability was measured based on propidium iodide (PI) exclusion after 5 days of rebound growth post-treatment (n = 3). (C), MOLM-13 cells were harvested after 15 hr of indicated treatments (Quiz: 3 nM quizartinib) to determine FLT3 activation by Western blotting. (D), MOLM-13 and MV4-11 cells were treated with either vehicle (V) or quizartinib (Q) for 48 hr in RPMI, hBMSC-CM, RPMI supplemented with heavy fraction (>30 kDa) of hBMSC-CM or light fraction (<30 kDa) of hBMSC-CM, followed by detection of apoptotic cells. (E), MOLM-13 cells were harvested after 16 hr of treatments as indicated and subjected to reverse-phase protein array analysis to explore the expression/activation levels of a wide range of proteins.

Figure 1—source data 1. Unedited raw blots for Figure 1C are shown.
Figure 1—source data 2. Unedited blots with labels for Figure 1C are shown.

Figure 1.

Figure 1—figure supplement 1. Human bone marrow stromal cells (hBMSC-CM) provide protection against apoptosis in FLT3-ITD acute myeloid leukemia (AML) cells following FLT3 inhibition by gilteritinib.

Figure 1—figure supplement 1.

(A, B) MOLM-13 cells were treated with either DMSO (veh) or 50 nM gilteritinib for 48 hr in RPMI or hBMSC-CM, followed by measurement of (A) apoptosis and (B) cell viability (n = 3).
Figure 1—figure supplement 2. Human bone marrow stromal cell (hBMSC-CM)-mediated protection does not coincide with alterations of the cell cycle.

Figure 1—figure supplement 2.

MOLM-13 cells were treated with the indicated conditions (Veh: DMSO; Quiz: 3 nM quizartinib) for 12 hr, followed by propidium iodide (PI) staining and cell cycle analysis via flow cytometry.
Figure 1—figure supplement 3. Human bone marrow stromal cell (hBMSC-CM) from HS-27A does not provide protection against apoptosis in FLT3-ITD acute myeloid leukemia (AML) cells following FLT3 inhibition.

Figure 1—figure supplement 3.

MOLM-13 cells were treated with the indicated conditions using conditioned media from HS-5 cells or HS-27A cells for 48 hr, followed by measurement of apoptosis.
Figure 1—figure supplement 4. Human bone marrow stromal cell (hBMSC-CM)-mediated protection is partially mimicked by BM-enriched cytokines.

Figure 1—figure supplement 4.

MOLM-13 cells were treated with either veh (V) or 3 nM quizartinib (Q) in the presence of RPMI, hBMSC-CM, and RPMI supplemented with 10 ng/mL IL-3 and/or 10 ng/mL GM-CSF, or 10 ng/mL FGF2 for 48 hr, followed by measurement of apoptosis.