*2.2. Ech A Inhibits the Activation of p38-MAPK and JNK in PB-CD34*<sup>+</sup> *Cells*

After 1 or 4 days of culture, the levels of phosphorylated p38-MAPK and JNK were significantly increased in the expanded cells (Figure 2A; negative control). However, the phosphorylation of p38-MAPK and JNK was dramatically suppressed upon the treatment of Ech A or NAC (Figure 2A). On the other hand, ERK1/2 phosphorylation remained unchanged in the cells. To examine whether p38-MAPK/JNK activation inhibits the ex vivo expansion of human PB-CD34<sup>+</sup> cells, cells were cultured in the presence of vehicle (0.1% DMSO), SB203580 (SB; 5 μM), or SP600125 (SP; 5 μM) for 1 or 4 days. The expanded cells were harvested for cell counts, and the analysis of CD34 expression by flow cytometry. As illustrated in Figure 2B, the total number of PBMCs was comparable irrespective of whether cells were incubated with or without SB or SP. Cell cultures with SB or SP showed a considerable increase in the number of CD34<sup>+</sup> cells compared to the DMSO control group. In particular, the number of CD34<sup>+</sup> cells was approximately 1.5-fold higher in the presence of SB (PBMCs, 2.17% <sup>±</sup> 0.06%, 1.45 <sup>±</sup> 0.05-fold; PB-CD34<sup>+</sup> cells, 72.27% <sup>±</sup> 5.01%, 6.38 <sup>±</sup> 0.27-fold) or SP (PBMCs, 2.33% <sup>±</sup> 0.31%, 1.51 <sup>±</sup> 0.22-fold; PB-CD34<sup>+</sup> cells, 71.27% <sup>±</sup> 7.83%, 6.34 <sup>±</sup> 0.61-fold) than that in the DMSO control group (PBMCs, 1.33% <sup>±</sup> 0.06%, 0.85 <sup>±</sup> 0.07-fold; PB-CD34<sup>+</sup> cells, 55.00% <sup>±</sup> 0.56%, 4.43 <sup>±</sup> 0.05-fold). However, ROS levels in PB-CD34<sup>+</sup> cells were unaffected by SB or SP (Figure 2C). These results suggest that p38-MAPK/JNK inhibition increases the ex vivo expansion of CD34<sup>+</sup> cells, even at high ROS levels.

**Figure 1.** Ech A increases PB-CD34<sup>+</sup> cell expansion by inhibiting reactive oxygen species' (ROS) generation. (**A**) Cells were treated with 10 μM 2 7 -dichlorodihydro-fluorescein diacetate for 30 min. The values in the 2 7 -dichlorofluorescein histograms indicate MFI (mean fluorescence intensity). (**B**,**C**) Cells were treated with 10 μM Ech A for 1 day (PB mononuclear cells, PBMCs) or 4 days (PB-CD34<sup>+</sup> cells). For NAC treatment, cells were treated with 5 mM NAC for 4 h, washed, suspended in complete medium, and incubated for an additional 1 day (PBMCs) or 4 days (PB-CD34<sup>+</sup> cells). (**B**) Total cell number was measured using the ADAM-MC automated mammalian cell counter (NanoEntech, Seoul, Korea). For flow cytometric immunophenotypic analysis, cells were stained with CD34-PE, CD38-FITC, CD45-APC, and 7-AAD. Each value was expressed as the mean ± SEM of three independent experiments. (**C**) Cells were treated with 10 μM 2 7 -dichlorodihydro-fluorescein diacetate for 30 min and ROS levels were subsequently measured usingthe flow cytometer.

**Figure 2.** Ech A enhances PB-CD34<sup>+</sup> cell expansion by suppressing the activation of p38-MAPK and JNK. Cells were treated with 10 μM Ech A for 1 day (PBMCs) or 4 days (PB-CD34<sup>+</sup> cells). For NAC treatment, cells were treated with 5 mM NAC for 4 h, washed, suspended in complete medium, and incubated for an additional 1 day (PBMCs) or 4 days (PB-CD34<sup>+</sup> cells). (**A**) Total protein was subjected to western blot analysis with the indicated antibodies. β-actin served as the loading control. (B, C) Cells were pretreated with SB203580 (10 μM) or SP600125 (10 μM) for 4 hr. (**B**) Total cell number was measured using the ADAM-MC automated mammalian cell counter. For flow cytometric immunophenotypic analysis, cells were stained with CD34-PE, CD38-FITC, CD45-APC, and 7-AAD. Each value was expressed as the mean ± SEM of three independent experiments. (**C**) Cells were treated with 10 μM 2 7 -dichlorodihydro-fluorescein diacetate for 30 min. The values in the 2 7 -dichlorofluorescein histograms indicate MFI.
