Supplementary Figure Legends
Supplementary Figure 1. ROS production is required for megakaryocytic differentiation of K562 and HEL cells. HEL and K562 cells were differentiated with 20 nM PMA in the presence or absence of 100 mM quercetin for 48h, and cell viability and proliferation were analysed. (A) Cell viability was analysed by annexin V-PE/7-AAD staining, the results show the percentage of viable cells (annexin V-PE/7-AAD double negative cells, means ± SD of 3 different experiments). (B) Cell proliferation was analysed by the MTT assay (means ± SD of 3 different experiments). (C) Intracellular ROS production in response to 20 nM PMA was measured by flow cytometry in K562 and HEL cells labelled with DCFDA 1h after PMA induction, in the presence or absence of 100µM NAC or 10 µM Trolox. ROS levels in response to PMA or PMA plus antioxidant were normalized to those of untreated cells or cells treated only with antioxidant, respectively. (D) HEL and K562 cells were differentiated with 20 nM PMA in the presence or absence of 100µM NAC or 10 µM Trolox. Cell staining with May-Grünwald-Giemsa of cells differentiated during 96h in the presence or absence of NAC or Trolox. Both antioxidants inhibited the acquisition of megakaryocytic morphological features. Differentiation was anlysed 48h after induction. (E) The levels of megakaryocytic markers (CD41 & CD61) in the presence of antioxidants plus PMA are lower than with PMA alone, whereas the level of the erythrocytic marker GpA is higher in the presence of antioxidants. A representative experiment in HEL cells treated with NAC and K562 treated with Trolox cells is shown. (F) NAC and Trolox treatment did not affect cell viability or cell proliferation (G) (means ± SD of 3 different experiments). (E) to (G) analysis were done after 48h of differentiation.
Supplementary Figure 2. The ROS burst is important for megakaryocytic differentiation. K562 and HEL cells were stimulated with 20 nM PMA. The PMA was washed-out 2, 4 or 6 h after the initial induction or maintained throughout the time of diffefentiation (48h). Differentiation was analysed 48h after the induction. Megakaryocytic marker levels (means ± SD of 3 different experiments) of HEL (A) and K562 (B) cells are shown. +++ p < 0.001, ++ p < 0.01 when compared to PMA-untreated control cells.
Supplementary Figure 3. Antioxidants hindered megakaryocytic differentiation of human CD34+ cells without affecting cell viability. Human CD34+ cells isolated from peripheral blood (cell purity was above 98%) were differentiated by treatment with 100 ng/ml TPO in the presence or absence of 10 mM Trolox or 100 mM NAC for 7 days. Cell viability was analysed by annexin V-PE/7-AAD staining, the results show the percentage of viable cells (annexin V-PE/7-AAD double negative cells, means ± SD of 3 different experiments).
Supplementary Figure 4. NADPH oxidase activity is required for megakaryocytic differentiation of K562 and HEL cells. (A) NADPH oxidase activity measured by extracellular superoxide (O2-) production in K562 stimulated by different concentrations of PMA (means ± SD of 3 different experiments performed in triplicate). (B) DPI treatment hindered megakaryocytic differentiation, a representative experiment in K562 cells 48h after induction is shown (please see also Figure 3). (C) DPI treatment did not affect cell viability, expressed as percentage of viable cells 48h after induction, (annexin V-PE/7-AAD double negative cells, means ± SD of 3 different experiments). (D) DPI treatment did not alter mitochondrial potential, monitored by the accumulation of the TMRE 48h after induction (means ± SD of 3 different experiments). (E) HEL and K562 cells were differentiated with 20 nM PMA in the presence or absence of 12.5 mM apocynin for 48h. Differentiation marker levels (means ± SD of 3 different experiments). (F) Cell viability represented as percentage of viable cells (annexin V-PE/7-AAD double negative cells, means ± SD of 3 different experiments). (G) Cell proliferation was analysed by the MTT assay (means ± SD of 3 different experiments).
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Supplementary Figure 5. Megakaryocytic differentiation dynamic of HEL and K562 cells. HEL and K562 cells were differentiated with 20 nM PMA in the presence or absence of 100 mM quercetin or 5 mM DPI. Differentiation marker levels were analysed at different time points (means ± SD of 3 different experiments). (A) Differentiation of HEL cells with or without quercetin. (B) Differentiation of HEL cells with or without DPI. (C) Differentiation of K562 cells with or without quercetin. (D) Differentiation of K562 cells with or without DPI.
Supplementary Figure 6. Down-regulation of p22phox protein affected megakaryocytic differentiation but not cell viability or proliferation. p22phox expression was down-regulated by RNAi experiments in HEL and K562 cells. A sequence directed against firefly luciferase was used as a control. Cells were differentiated with 20 nM PMA for 48h as in figure 4. (A) Cell viability was analysed by annexin V-PE/7-AAD staining, the results show the percentage of viable cells (annexin V-PE/7-AAD double negative cells, means ± SD of 3 different experiments). (B) Cell proliferation was analysed by the MTT assay (means ± SD of 3 different experiments).
Supplementary Figure 7. Analysis of the signalling pathways activated during megakaryocytic differentiation of K562 and HEL cells. Cells were treated with 20 nM PMA or 100 ng/ml TPO. Samples were harvested at different times and the expression of the phosphorylated/active forms of ERK, JNK, AKT, STAT3 and STAT5 was analysed by immunoblotting using specific antibodies. The same membranes were stripped and reprobed to check the total levels of these proteins. (A) Expression of phophorylated ERK and JNK forms in K562 and HEL cells treated with PMA (B). Expression of AKT, STAT3 and STAT5 phophorylated forms in HEL cells treated with TPO.
Supplementary Figure 8. Analysis of signalling pathways involved in the megakaryocytic differentiation. K562 and HEL cells were differentiated with 20 nM PMA during 48h. Cell differentiaton was performed in the presence or absence of specific inhibitors for MEK (10 µM U0126), JNK (10 µM SP600125), PI3K (10 µM LY294002), AKT (10 µM AKT Inhibitor X) and JAK2 (50 µM AG490). Cell viability (A) and proliferation (B) were analysed. The means ± SD of 3 different experiments are shown.