Design of Hoxa3-mCherry and mCherry plasmids and expression in HSC/Ps. (A) Schematic of signal peptide (SP)–Hoxa3-mCherry and SP-mCherry constructs, driven by the cytomegalovirus promoter (P CMV). (B) SP-mCherry, and SP-Hoxa3-mCherry, protein expression in AFT024 feeder layer cells, shown at original magnification × 200. (C) Western blot detection of mCherry in nuclear and cytoplasmic protein extraction of HSC/Ps cocultured with AFT024 cells secreting SP-Hoxa3-mCherry protein (predicted size = 73.17 kDa) or SP-mCherry control (predicted size = 26.73 kDa). (D) Immunofluorescent detection of mCherry in hematopoietic progenitor cells purified from coculture with AFT024/SP-mCherry feeder layer or AFT024/SP-Hoxa3-mCherry feeder layer. DAPI was used to label nuclei. Image is original magnification ×600.