*2.3. Ech A Regulates PB-CD34*<sup>+</sup> *Cell Expansion via PI3K*/*Akt Pathway*

We further assessed the possible molecular mechanisms underlying Ech A-mediated ex vivo expansion of PB-CD34<sup>+</sup> cells. Our results showed that phosphorylation of PI3K-p85 and Akt was enhanced to a greater extent in Ech A- or NAC-treated cells than that in untreated cells (Figure 3A); however, phospho-PTEN level were reduced. To verify the requirement of PI3K/Akt signaling activation for PB-CD34<sup>+</sup> cell expansion, we used the PI3K/Akt inhibitor LY294002. As shown in Figure 3B, pretreatment of the Ech A or NAC treatment group with LY294002 (PBMCs, 7.83 ± 1.31%, 3.29 <sup>±</sup> 0.73-fold; PB-CD34<sup>+</sup> cells, 71.43 <sup>±</sup> 6.45%, 9.55 <sup>±</sup> 1.41-fold) substantially reduced the percentage of CD34<sup>+</sup> cells as well as CD34<sup>+</sup> cell number compared to Ech A or NAC single treatment group (PBMCs, 23.63 <sup>±</sup> 1.7%, 14.43 <sup>±</sup> 2.42-fold; PB-CD34<sup>+</sup> cells, 84.43 <sup>±</sup> 0.93%, 22.33 <sup>±</sup> 1.49-fold).

**Figure 3.** Echinochrome A (Ech A) upregulates PB-CD34<sup>+</sup> cell expansion by activating PI3K/Akt pathway. 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 cell lysates for each condition were immunoblotted with the indicated antibodies. (**B**) Cell were pretreated with LY294002 (10 μM) for 4 h. Total cell number was determined 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.
