Figure 5.
Microtubule inhibition blocks mitochondrial transfer and releases ALL cells from ROS-induced, MSC-mediated protection. (Ai) Agarose gel with PCR products from amplification of HS27a mitochondrial DNA, with or without mitochondrial depletion. (ii) Fluorescence microscopy imaging of MitoTracker dye in HS27a cells, with or without mitochondrial depletion. (iii) Imaging (original magnification ×40) of mitochondrially depleted (mito-depleted) HS27a cells in culture with SEM cells after phalloidin and DAPI staining. (iv) Percentage of apoptosis (annexin V+, DAPI−, y-axis) of SEM cells treated with AraC, SEM cells cocultured with HS27a treated with AraC, or SEM cocultured with HS27a mito-depleted cells treated with AraC (x-axis). All statistically significant comparisons (by unpaired Student t test) are as depicted: HS27a vs mito-depleted HS27a cells, P = .0008. (v) Percentage cell death (DAPI+, y-axis) of SEM cells treated with AraC, cocultured with HS27a treated with AraC, or cocultured with HS27a mito-depleted cells treated with AraC (x-axis). All statistically significant comparisons (by unpaired Student t test) are as depicted: MH27a vs mito-depleted HS27a, P = .0043. (vi) Percentage cell death or apoptosis (DAPI+ or annexin V+/DAPI−, y-axis) of SEM cells+AraC, SEM cells cocultured with HS27a cells+AraC, or SEM cells cocultured with mito-depleted HS27a+AraC (x-axis). All data are the mean ± SE of results in 3 independent experiments. All statistically significant comparisons (by unpaired Student t test) are as depicted: MH27a vs mito-depleted HS27a cells, P < .0001. (B) Mitochondrial transfer from HS27a to SEM cells after microtubule-damaging blockade. (MitoTracker MFI, y-axis). The baseline condition is coculture with no added agents; all other conditions are AraC treated, either alone or with latrunculin-B (lat-B) and nocodazole (nocod) (x-axis). All statistically significant comparisons (by unpaired Student t test) are as depicted: none vs AraC, P = .0005; AraC vs AraC+lat-B, P = .0028; and AraC vs AraC+nocod, P < .0001. (Ci) Percentage viability (y-axis) after treatment of SEM ALL cells with the agents indicated (x-axis). (ii) Relative viability (y-axis) after treatment of HS27a cells with the agents indicated (x-axis). (iii) Percentage cell death (y-axis) after AraC-treatment of SEM, either in monoculture or coculture with HS27a cells, alone or with lat-B+nocod, colchicine, or VCR added (x-axis). All data are the mean ± SE of 3 independent experiments. All statistically significant comparisons (by unpaired Student t test) are as depicted: MSC none vs lat-B, P = .0004; MSC none vs nocod, P = .0018; MSC none vs colchicine, P = .0167; and MSC none vs VCR, P = .0002. (iv) Phalloidin and DAPI staining of HS27a (original magnification ×40) after exposure to nocodazole or colchicine. *.01 < P ≤ .05; **.001 < P ≤ .01; ***.0001 < P ≤ .001; ****P ≤ .0001.

Microtubule inhibition blocks mitochondrial transfer and releases ALL cells from ROS-induced, MSC-mediated protection. (Ai) Agarose gel with PCR products from amplification of HS27a mitochondrial DNA, with or without mitochondrial depletion. (ii) Fluorescence microscopy imaging of MitoTracker dye in HS27a cells, with or without mitochondrial depletion. (iii) Imaging (original magnification ×40) of mitochondrially depleted (mito-depleted) HS27a cells in culture with SEM cells after phalloidin and DAPI staining. (iv) Percentage of apoptosis (annexin V+, DAPI−, y-axis) of SEM cells treated with AraC, SEM cells cocultured with HS27a treated with AraC, or SEM cocultured with HS27a mito-depleted cells treated with AraC (x-axis). All statistically significant comparisons (by unpaired Student t test) are as depicted: HS27a vs mito-depleted HS27a cells, P = .0008. (v) Percentage cell death (DAPI+, y-axis) of SEM cells treated with AraC, cocultured with HS27a treated with AraC, or cocultured with HS27a mito-depleted cells treated with AraC (x-axis). All statistically significant comparisons (by unpaired Student t test) are as depicted: MH27a vs mito-depleted HS27a, P = .0043. (vi) Percentage cell death or apoptosis (DAPI+ or annexin V+/DAPI−, y-axis) of SEM cells+AraC, SEM cells cocultured with HS27a cells+AraC, or SEM cells cocultured with mito-depleted HS27a+AraC (x-axis). All data are the mean ± SE of results in 3 independent experiments. All statistically significant comparisons (by unpaired Student t test) are as depicted: MH27a vs mito-depleted HS27a cells, P < .0001. (B) Mitochondrial transfer from HS27a to SEM cells after microtubule-damaging blockade. (MitoTracker MFI, y-axis). The baseline condition is coculture with no added agents; all other conditions are AraC treated, either alone or with latrunculin-B (lat-B) and nocodazole (nocod) (x-axis). All statistically significant comparisons (by unpaired Student t test) are as depicted: none vs AraC, P = .0005; AraC vs AraC+lat-B, P = .0028; and AraC vs AraC+nocod, P < .0001. (Ci) Percentage viability (y-axis) after treatment of SEM ALL cells with the agents indicated (x-axis). (ii) Relative viability (y-axis) after treatment of HS27a cells with the agents indicated (x-axis). (iii) Percentage cell death (y-axis) after AraC-treatment of SEM, either in monoculture or coculture with HS27a cells, alone or with lat-B+nocod, colchicine, or VCR added (x-axis). All data are the mean ± SE of 3 independent experiments. All statistically significant comparisons (by unpaired Student t test) are as depicted: MSC none vs lat-B, P = .0004; MSC none vs nocod, P = .0018; MSC none vs colchicine, P = .0167; and MSC none vs VCR, P = .0002. (iv) Phalloidin and DAPI staining of HS27a (original magnification ×40) after exposure to nocodazole or colchicine. *.01 < P ≤ .05; **.001 < P ≤ .01; ***.0001 < P ≤ .001; ****P ≤ .0001.

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