Figure 1.
The optimization of the PMA-induced megakaryocytic differentiation in K562 cells.
(A) K562 cells were incubated with 5 nM PMA for the indicated time and then the expression of CD41 and CD61 were analyzed by flow cytometry. (B) Cell morphology was observed by microscope. Scale bars represent 50 µm. (C) Megakaryocytic differentiation was detected by modified Wright-Giemsa staining for cell morphology. Representative cytological changes at 72 h, such as increase in nuclear-to-cytoplasm ratio (a), larger cells (b), and polylobulation nucleus (c) were denoted. Scale bars represent 50 µm. (D) K562 cells were induced with different concentration PMA for 72 h. Cell apoptosis were detected by flow cytometry. All graphics represented means ± SD obtained from three independent experiments, *p≤0.05, **p≤0.001.
Figure 2.
Alterations in mitochondrial functions occured during PMA-induced K562 differentiation.
1×106 K562 cells were incubated with 5 nM PMA and harvested at 12 h, 24 h and 72 h respectively, stained with ROS fluorescence indicator MitoSOX (A), Fluo-3AM (B), and JC-1(C), followed by flow cytometry analysis. Data represented means ± SD obtained from three independent experiments, *p≤0.05. (D) O2 consumption by K562 cells during megakaryocytic differentiation were analyzed by ESR. (a): The K parameter curve. (b): Kinetic curve of O2 uptake by K562 cells. Graphics represented means ± SD obtained from three independent experiments. (E) Intracellular ATP level was measured using luminescence ATP detection assay system. Data represented means ± SD obtained from three independent experiments, *p≤0.05.
Figure 3.
Effects of PMA-induced megakaryocytic differentiation on mitochondrial morphology and ultrastructure.
1×106 K562 cells were incubated with 5 nM PMA and harvested at 12 h, 24 h and 72 h respectively, Cells labeled with NAO were detected by flow cytometry analysis and confocal microscopy (A, B). Scale bars represent 5 µm. Data were shown as mean ± SD of three independent experiments, * p≤0.05. (C) Mitochondria ultrastructure was analyzed by transmission electron microscopy. Scale bars represent 2 µm in the left panels and 100 nm in the right panels. The bracketed regions in the left panels are enlarged in the right panels.
Figure 5.
PMA induced K562 cells differentiation reduced the activity of respiratory chain complex IV.
(A) K562 cells were incubated with 5 nM PMA and harvested at 12 h, 24 h and 72 h respectively. Isolated mitochondria from these cells were solubilised with 1% dodecyl-maltoside (DM) before subjecting the samples to hrCN-PAGE. After electrophoresis, gels were incubated with in-gel catalytic activity assay buffers. Each lane was loaded with 100 µg protein. (B) K562 cells were incubated with 5 nM PMA and harvested at 12 h, 24 h and 72 h respectively. The activity of the complex IV activity was detected by spectrophotometric measurement method. Data were shown as mean ± SD of three independent experiments, *p≤0.05, **p≤0.001. (C) K562 cells were incubated with 5 nM PMA for the indicated time. Mitochondrial extracts were prepared and subjected to SDS-PAGE followed by immunoblotting with antibodies indicated. (D) Mitochondrial extracts were prepared and subjected to Blue Native gel subsequently processed by immunoblotting to analyze the levels of the four subunits (COX1, COX3, COX5A and COX6B1) of complex IV. The arrows represented the complex of interest. Blots were representative of three separate experiments.
Figure 4.
Stability of mitochondrial membrane potential promoted the PMA-induced K562 cell differentiation.
(A) K562 cells were pre-treated with different doses of CsA for 6 h. After the supernatant was replaced by fresh medium without CsA, cells differentiation was induced by 5 nM PMA for 72 h. The mitochondrial membrane potential (JC-1 staining) was determined by flow cytometry analysis. (B) K562 cells were pre-treated with 5 µM CsA for 6 h, and then induced by 5 nM PMA and harvested at 12 h, 24 h and 72 h respectively. Mitochondrial membrane potential (JC-1 staining) was determined by flow cytometry analysis. (C & D) After pre-treatment by 5 µM CsA, K562 cells were induced using 2 nM or 5 nM PMA for 72 h, expression of CD41 and CD61was determined by flow cytometry analysis. All graphics represented means ± SD obtained from three independent experiments, *p≤0.05, **p≤0.001.
Figure 6.
Inhibition of respiratory chain complex IV activity decreased mitochondrial membrane potential.
(A) K562 cells were pre-treated with different doses of SA for 3 hour, and then the mitochondrial membrane potential (JC-1 staining) was determined by flow cytometry analysis. (B) K562 cells (3×105 cells/ml) were pre-treated with 5 µM CsA for 6 h and then treated with 5 nM PMA for the indicated time. The mitochondrial protein complexes were separated by hrCN-PAGE gel and then identified by in-gel catalytic activity assay of complex IV. (C & D) K562 cells were pre-treated with the complex IV specific inhibitor SA for 3 h, then cells were induced by 5 nM PMA for 72 h. Expression of CD41 and CD61 was determined by flow cytometry analysis. Data were shown as mean ± SD of three independent experiments, *p≤0.05, **p≤0.001.