Figure 4.
AraC and DNR stimulate mitochondrial transfer from healthy donor or HS27a MSCs to ALL cells via TNTs. (A) Phalloidin and DAPI staining of SEM+HD MSC coculture incubated with AraC or VCR at original magnifications ×10 and ×40. Red arrows indicate SEM cells (round with prominent nuclei) in physical contact with MSCs. (Bi) Mitochondrial transfer by MitoTracker assay (mean fluorescence intensity [MFI], y-axis), from HS27a to SEM, TOM1, and SD1 ALL cells in contact or in a Transwell (x-axis). All data are the mean ± SE of 3 independent experiments. All statistically significant comparisons (by unpaired Student t test) are as depicted: SEM HS27a vs HS27a Transwell, P < .0001; TOM1 HS27a vs Transwell, P < .0001; and SD1 HS27a vs Transwell, P < .0001. (ii) Mitochondrial transfer by MitoTracker assay (MFI, y-axis), from healthy donor MSC to healthy donor B-cells (3 independent experiments) or patient ALL cells identified by UKALL14 trial number (UPN; x-axis). (C) Mitochondrial transfer by MitoTracker assay (MFI, y-axis) from HS27a to SEM after coculture and either no treatment or treatment with AraC, VCR, or DEX. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs AraC, P < .0001; AraC vs DEX, P < .0001; and AraC vs VCR, P = .0003. (Di) Mitochondrial transfer by MitoTracker assay (MFI, y-axis) of SEM cells in coculture with HS27a MSCs after no treatment, AraC, or 5 mM AraC+NAC. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs AraC, P < .0001, and AraC vs AraC+NAC, P < .0001. (ii) Mitochondrial mass by MitoTracker assay (MFI, y-axis) of SEM cells in coculture with HS27a MSCs after no treatment, DNR or 5 mM DNR+NAC. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs DNR, P = .0002, and DNR vs DNR+NAC, P = .0002. (iii) Mitochondrial mass by MitoTracker assay of REH cells in coculture with HS27a MSCs after no treatment, AraC, or 5 mM AraC+NAC. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs AraC, P < .0001, and AraC vs AraC+NAC, P < .0001. (E) Live-cell confocal imaging of HS27a cells, stained deep red with MitoTracker in coculture with SEM ALL cells stained with DiO. Images were taken at the time points indicated (3 minutes apart). The blue and green arrows each indicate the progression of 2 individual mitochondria along a TNT. (F) Agarose gel images showing PCR products from human nuclear and mitochondrial DNA and murine nuclear and mitochondrial DNA, as indicated in each quadrant. Lane 1, MS-5 murine MSCs; lane 2, SEM cells; lanes 3 to 5, SEM cells sorted after coculture with MS-5; lanes 6 to 8, SEM cells sorted after AraC-treated coculture with MS-5. Human nuclear DNA PCR in lane 5 failed. ***.0001 < P ≤ .001; ****P ≤ .0001.

AraC and DNR stimulate mitochondrial transfer from healthy donor or HS27a MSCs to ALL cells via TNTs. (A) Phalloidin and DAPI staining of SEM+HD MSC coculture incubated with AraC or VCR at original magnifications ×10 and ×40. Red arrows indicate SEM cells (round with prominent nuclei) in physical contact with MSCs. (Bi) Mitochondrial transfer by MitoTracker assay (mean fluorescence intensity [MFI], y-axis), from HS27a to SEM, TOM1, and SD1 ALL cells in contact or in a Transwell (x-axis). All data are the mean ± SE of 3 independent experiments. All statistically significant comparisons (by unpaired Student t test) are as depicted: SEM HS27a vs HS27a Transwell, P < .0001; TOM1 HS27a vs Transwell, P < .0001; and SD1 HS27a vs Transwell, P < .0001. (ii) Mitochondrial transfer by MitoTracker assay (MFI, y-axis), from healthy donor MSC to healthy donor B-cells (3 independent experiments) or patient ALL cells identified by UKALL14 trial number (UPN; x-axis). (C) Mitochondrial transfer by MitoTracker assay (MFI, y-axis) from HS27a to SEM after coculture and either no treatment or treatment with AraC, VCR, or DEX. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs AraC, P < .0001; AraC vs DEX, P < .0001; and AraC vs VCR, P = .0003. (Di) Mitochondrial transfer by MitoTracker assay (MFI, y-axis) of SEM cells in coculture with HS27a MSCs after no treatment, AraC, or 5 mM AraC+NAC. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs AraC, P < .0001, and AraC vs AraC+NAC, P < .0001. (ii) Mitochondrial mass by MitoTracker assay (MFI, y-axis) of SEM cells in coculture with HS27a MSCs after no treatment, DNR or 5 mM DNR+NAC. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs DNR, P = .0002, and DNR vs DNR+NAC, P = .0002. (iii) Mitochondrial mass by MitoTracker assay of REH cells in coculture with HS27a MSCs after no treatment, AraC, or 5 mM AraC+NAC. All statistically significant comparisons (by unpaired Student t test) are as depicted: no treatment vs AraC, P < .0001, and AraC vs AraC+NAC, P < .0001. (E) Live-cell confocal imaging of HS27a cells, stained deep red with MitoTracker in coculture with SEM ALL cells stained with DiO. Images were taken at the time points indicated (3 minutes apart). The blue and green arrows each indicate the progression of 2 individual mitochondria along a TNT. (F) Agarose gel images showing PCR products from human nuclear and mitochondrial DNA and murine nuclear and mitochondrial DNA, as indicated in each quadrant. Lane 1, MS-5 murine MSCs; lane 2, SEM cells; lanes 3 to 5, SEM cells sorted after coculture with MS-5; lanes 6 to 8, SEM cells sorted after AraC-treated coculture with MS-5. Human nuclear DNA PCR in lane 5 failed. ***.0001 < P ≤ .001; ****P ≤ .0001.

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