*2.5. Src*/*Lyn Is a Major Upstream Signal for Ech A-Induced p110*δ*-Mediated CD34*<sup>+</sup> *Cell Expansion*

We also investigated Src family kinases associated with the increased expression of p110δ upon Ech A or NAC treatment in PB-CD34<sup>+</sup> cells. Compared with untreated controls, treatment with Ech A or NAC increased Src phosphorylation levels, but produced little effect on Fyn phosphorylation. Interestingly, Lyn was significantly phosphorylated only by Ech A treatment, but not by NAC (Figure 5A). To identify the requirement for the Src/Lyn pathway in Ech A- or NAC-induced p110δ expression, we used the Src/Lyn inhibitor PP1. The percentages of CD34<sup>+</sup> cells and the CD34+ cell number in the Ech A or NAC single treatment group (PBMCs, 14.2% <sup>±</sup> 1.39%, 8.37 <sup>±</sup> 0.13-fold; PB-CD34<sup>+</sup> cells, 81.57% ± 1.88%, 9.43 ± 0.55-fold) were significantly lower than that in the PP1 pretreatment group (PBMCs, 8.63% <sup>±</sup> 2.65%, 4.41 <sup>±</sup> 1.40-fold; PB-CD34<sup>+</sup> cells, 70.27% <sup>±</sup> 1.54%, 6.84 <sup>±</sup> 0.11-fold) (Figure 5B). To establish a possible link between Ech A- and NAC-induced Src/Lyn phosphorylation and PI3K activation during the ex vivo expansion of CD34<sup>+</sup> cells, we treated cells with PP1, LY294002, or CAL-101. First, we confirmed the activity of various inhibitors used in this study on each target molecule (Figure S3). As shown in Figure 5C, PP1 dramatically suppressed Ech A or NAC-mediated expression of p110δ and PI3K/Akt activation. On the other hand, LY294002 and CAL-101 did not inhibit Ech A- or NAC-induced Src/Lyn activation (Figure 5D,E). These results suggest that Lyn is activated prior to p110δ and PI3K/Akt and plays a critical role as an upstream regulatory molecule in Ech A-induced ex vivo expansion of PB-CD34<sup>+</sup> cells.

**Figure 5.** Src/Lyn is major upstream signal for p110δ-mediated CD34<sup>+</sup> cell expansion by Ech A. 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**,**C**–**E**) Total cell lysates for each condition were harvested and immunoblotted with the indicated antibodies. (**B**–**E**) Cells were pretreated with PP1 (10 μM), LY294002 (10 μM), or CAL-101 (20 μM) for 4 h. (**B**) 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.
