American Society of Hematology

Figure 1

$Figure 1. AML cells adherent to bone marrow mesenchymal cells have a survival selective advantage during in vitro cytarabine treatment associated with exogenous mitochondrial marker intake. (A) Pie charts showing the grand mean percentage determined from independent experiments of live adherent (A) and nonadherent cells (NA) for AML cell lines (HL60, Kasumi-1, KG-1, MOLM-14, NB4, SKM-1, THP1, and U937) or primary samples (n = 3) in CT and ARA conditions. (B) A or NA fractions of the specified AML cell lines were collected separately after 72 hours of coculture with MS-5 with or without ARA at 3 µM. Data represent the percentage of viable (Annexin-V−, DAPI−) AML cells (SSC-low, GFP+, SCA-1-PE−) for A and NA fractions in the untreated (CT) and ARA condition. n = 3 per cell line. **P < .01 by ANOVA. (C) (Left) Representative example of FCM analysis of MitoTracker (MT) uptake by Kasumi-1 AML cells (SSC-low, GFP+) cocultured for 0, 6, or 72 hours with MitoTracker Red–loaded MS-5 cells. (Middle) Gray dashed line represents the MT fluorescence intensity of MS-5 cells (SSC-high, GFP−). Solid lines represent the MT fluorescence intensity of Kasumi-1 cells at 0 hours (black) or after 6 (pink) or 72 hours (red) of coculture. (Right) MT fluorescence intensity for A (red) or NA (blue) Kasumi-1 cells. (D) eGFP-AML cell lines were cocultured with MitoTracker Red–loaded MS-5 cells in contact or in Transwell plate noncontact coculture for 72 hours. Data show the MitroTracker red grand mean of median fluorescence intensity (MFI; n = 3 per cell line) for the GFP+ gated cells. NSP > .05, ***P < .001, by ANOVA. (E) MS-5 stably expressing OMI/HTRA2-mCherry was treated with 0.3 or 1 µM STS for 18 hours. Data show mCherry fluorescence intensity (upper) or the mitochondrial membrane potential using TMRE (lower) of MS-5 OMI/HTRA2-mCherry cells in untreated (CT; red lines) and STS conditions (black dashed or dotted lines). (F) Representative FACS plots showing the mCherry and GFP fluorescence of isolated MS-5 OMI/HTRA2-mCherry (top), isolated MOLM-14 GFP (middle), and coculture (bottom). Red gate shows the mCherry fluorescence apparition for the MOLM-14 GFP+ AML cells after 72 hours of coculture. (G) Column scatter plot shows the percentage GFP+mCherry+ live cells in the GFP+ gated population of the specified AML cell lines (n = 3 per cell line) after 24 hours of coculture with MS-5 OMI/HTRA2-mCherry with the indicated dose of STS normalized to control condition.$

AML cells adherent to bone marrow mesenchymal cells have a survival selective advantage during in vitro cytarabine treatment associated with exogenous mitochondrial marker intake. (A) Pie charts showing the grand mean percentage determined from independent experiments of live adherent (A) and nonadherent cells (NA) for AML cell lines (HL60, Kasumi-1, KG-1, MOLM-14, NB4, SKM-1, THP1, and U937) or primary samples (n = 3) in CT and ARA conditions. (B) A or NA fractions of the specified AML cell lines were collected separately after 72 hours of coculture with MS-5 with or without ARA at 3 µM. Data represent the percentage of viable (Annexin-V⁻, DAPI⁻) AML cells (SSC-low, GFP⁺, SCA-1-PE⁻) for A and NA fractions in the untreated (CT) and ARA condition. n = 3 per cell line. **P < .01 by ANOVA. (C) (Left) Representative example of FCM analysis of MitoTracker (MT) uptake by Kasumi-1 AML cells (SSC-low, GFP+) cocultured for 0, 6, or 72 hours with MitoTracker Red–loaded MS-5 cells. (Middle) Gray dashed line represents the MT fluorescence intensity of MS-5 cells (SSC-high, GFP⁻). Solid lines represent the MT fluorescence intensity of Kasumi-1 cells at 0 hours (black) or after 6 (pink) or 72 hours (red) of coculture. (Right) MT fluorescence intensity for A (red) or NA (blue) Kasumi-1 cells. (D) eGFP-AML cell lines were cocultured with MitoTracker Red–loaded MS-5 cells in contact or in Transwell plate noncontact coculture for 72 hours. Data show the MitroTracker red grand mean of median fluorescence intensity (MFI; n = 3 per cell line) for the GFP⁺ gated cells. ^NSP > .05, ***P < .001, by ANOVA. (E) MS-5 stably expressing OMI/HTRA2-mCherry was treated with 0.3 or 1 µM STS for 18 hours. Data show mCherry fluorescence intensity (upper) or the mitochondrial membrane potential using TMRE (lower) of MS-5 OMI/HTRA2-mCherry cells in untreated (CT; red lines) and STS conditions (black dashed or dotted lines). (F) Representative FACS plots showing the mCherry and GFP fluorescence of isolated MS-5 OMI/HTRA2-mCherry (top), isolated MOLM-14 GFP (middle), and coculture (bottom). Red gate shows the mCherry fluorescence apparition for the MOLM-14 GFP⁺ AML cells after 72 hours of coculture. (G) Column scatter plot shows the percentage GFP⁺mCherry⁺ live cells in the GFP⁺ gated population of the specified AML cell lines (n = 3 per cell line) after 24 hours of coculture with MS-5 OMI/HTRA2-mCherry with the indicated dose of STS normalized to control condition.

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