*2.4. The PI3K p110*δ *Isoform Is Required for Ech A-Induced CD34*<sup>+</sup> *Cell Expansion*

Next, we examined which p110 isoforms were associated with the expansion of PB-CD34<sup>+</sup> cells, following Ech A or NAC treatment using antibodies, each specific for different p110 isoforms of PI3K. Ech A- or NAC-treated PBMCs or PB-CD34<sup>+</sup> cells showed a higher expression of p110δ than untreated PB-CD34<sup>+</sup> cells; however, p110α, p110β, and p110γ expression was not affected by Ech A or NAC treatment (Figure 4A). To identify the role of p110δ in Ech A- or NAC-induced PB-CD34<sup>+</sup> cell expansion, cells were treated with Ech A or NAC in the presence or absence of the p110δ specific inhibitor, CAL-101. The percentage of CD34<sup>+</sup> cells and the CD34<sup>+</sup> cell number in the Ech A or NAC single treatment group (PBMCs, 23.63% <sup>±</sup> 1.7%, 15.32 <sup>±</sup> 1.27-fold; PB-CD34<sup>+</sup> cells, 82.07% <sup>±</sup> 0.32%, 18.55 <sup>±</sup> 1.12-fold) were remarkably decreased by CAL-101 pretreatment (PBMCs, 7.57% <sup>±</sup> 1.99%, 3.37 <sup>±</sup> 0.74-fold; PB-CD34<sup>+</sup> cells, 70.67% ± 2.73%, 7.93 ± 1.23-fold) (Figure 4B).

**Figure 4.** Ech A induces the activation of the PI3K p110δ isoform for PB-CD34<sup>+</sup> cell expansion. 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. (**B**) Cells were pretreated with CAL-101 (20 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.
