Figure 4.
SLFN14-deficient MKs show a mitochondrial defect. (A) Transmission EM imaging of differentiation day 3 MK cells shows abnormal mitochondria in SLFN14-deficient MK compared with WT/WT MK. Images taken after a blinded analysis of MK at passage week 17 for WT/WT and WT/K219N and week 3 for K219N/K219N. (B) Confocal imaging of day 4 MK cells from WT and mutant imMKCL with MitoTracker Red to visualize (top) and quantify (bottom) mitochondria. Imaging performed at passage week 6 for WT/K219N and K219N/K219N, and week 13 for WT/WT. Quantification of the mean number (nr) of branches per mitochondrial network (bottom left) and mean mitochondrial branch length (bottom right) as calculated by the Mitochondrial Network Analysis software for 25 randomly selected MK containing a single nucleus for each condition. P values were determined using the pairwise Wilcoxon test for all comparisons. (C) Confocal imaging of day 8 MK from blood-derived stem cells using the ATP5E antibody (green) to visualize (top) and quantify (bottom) mitochondria. Phalloidin staining in red. Quantification of the mean number of branches per mitochondrial network (bottom) as calculated by the Mitochondrial Network Analysis software for 25 randomly selected MK containing a single nucleus for each condition. P values were determined using the Wilcoxon test. (D) OCR measurements were obtained over time (minutes) using the extracellular flux analyzer from Seahorse Bioscience for WT/WT, WT/K219N, and K219N/K219N MK on day 4 (left). Values represent the mean and standard deviation for each condition of the Seahorse assays obtained for 4 replicates from 2 differentiation experiments. The mitochondrial stress test was used to obtain diverse parameters: ATP-linked OCR (by adding the ATP synthase inhibitor oligomycin), maximal OCR (by adding the uncoupling agent FCCP), and complete inhibition of the mitochondria (by adding the inhibitor antimycin) (right top). The maximal respiration capacity and ATP-linked respiration were significantly reduced for WT/K219N and K219N/K219N MK compared with WT/WT MK (right). Statistics using a pairwise Wilcoxon test was performed. Boxplots in all panels represent the middle 50% of the data points with, 25% outliers as whiskers and the mean as a black horizontal line. All individual data points were added as dots to the graph. (E) Representative gel electrophoresis image showing rRNA degradation fragments (red arrows) for day 4 differentiated WT/K219N and K219N/K219N MK that are not present in WT/WT MK or undifferentiated cells (left). Experiments performed at passage week 5 for day 0 and week 7 for day 4. The concentration of total RNA (ng/μL) for the different samples was added below the figure. Quantification of the RIN values obtained for RNA samples of imMKCL cells from 4 (day 0) and 9 (day 4) independent differentiation experiments on days 0 and 4 WT/WT, WT/K219N, and K219N/K219N MK (multiple technical replicates included per time point per condition) (right). Experiments performed at passage weeks 2, 3, 4, 5, 7, 9, 12, 23, and 25. Boxplots represent the middle 50% of the data points with, 25% outliers as whiskers and the mean as a black horizontal line. All individual data points were added as dots to the graph. Statistical analyses were performed using the pairwise Wilcoxon test for all comparisons. Scale bars, 1 μm (A) and 10 μm (B-C).

SLFN14-deficient MKs show a mitochondrial defect. (A) Transmission EM imaging of differentiation day 3 MK cells shows abnormal mitochondria in SLFN14-deficient MK compared with WT/WT MK. Images taken after a blinded analysis of MK at passage week 17 for WT/WT and WT/K219N and week 3 for K219N/K219N. (B) Confocal imaging of day 4 MK cells from WT and mutant imMKCL with MitoTracker Red to visualize (top) and quantify (bottom) mitochondria. Imaging performed at passage week 6 for WT/K219N and K219N/K219N, and week 13 for WT/WT. Quantification of the mean number (nr) of branches per mitochondrial network (bottom left) and mean mitochondrial branch length (bottom right) as calculated by the Mitochondrial Network Analysis software for 25 randomly selected MK containing a single nucleus for each condition. P values were determined using the pairwise Wilcoxon test for all comparisons. (C) Confocal imaging of day 8 MK from blood-derived stem cells using the ATP5E antibody (green) to visualize (top) and quantify (bottom) mitochondria. Phalloidin staining in red. Quantification of the mean number of branches per mitochondrial network (bottom) as calculated by the Mitochondrial Network Analysis software for 25 randomly selected MK containing a single nucleus for each condition. P values were determined using the Wilcoxon test. (D) OCR measurements were obtained over time (minutes) using the extracellular flux analyzer from Seahorse Bioscience for WT/WT, WT/K219N, and K219N/K219N MK on day 4 (left). Values represent the mean and standard deviation for each condition of the Seahorse assays obtained for 4 replicates from 2 differentiation experiments. The mitochondrial stress test was used to obtain diverse parameters: ATP-linked OCR (by adding the ATP synthase inhibitor oligomycin), maximal OCR (by adding the uncoupling agent FCCP), and complete inhibition of the mitochondria (by adding the inhibitor antimycin) (right top). The maximal respiration capacity and ATP-linked respiration were significantly reduced for WT/K219N and K219N/K219N MK compared with WT/WT MK (right). Statistics using a pairwise Wilcoxon test was performed. Boxplots in all panels represent the middle 50% of the data points with, 25% outliers as whiskers and the mean as a black horizontal line. All individual data points were added as dots to the graph. (E) Representative gel electrophoresis image showing rRNA degradation fragments (red arrows) for day 4 differentiated WT/K219N and K219N/K219N MK that are not present in WT/WT MK or undifferentiated cells (left). Experiments performed at passage week 5 for day 0 and week 7 for day 4. The concentration of total RNA (ng/μL) for the different samples was added below the figure. Quantification of the RIN values obtained for RNA samples of imMKCL cells from 4 (day 0) and 9 (day 4) independent differentiation experiments on days 0 and 4 WT/WT, WT/K219N, and K219N/K219N MK (multiple technical replicates included per time point per condition) (right). Experiments performed at passage weeks 2, 3, 4, 5, 7, 9, 12, 23, and 25. Boxplots represent the middle 50% of the data points with, 25% outliers as whiskers and the mean as a black horizontal line. All individual data points were added as dots to the graph. Statistical analyses were performed using the pairwise Wilcoxon test for all comparisons. Scale bars, 1 μm (A) and 10 μm (B-C).

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