Blood test and colony forming cell (CFC) assay

Peripheral blood samples were isolated from mutants and WT littermates and collected into Vacutainer plastic tubes coated with K₂EDTA. An automated complete blood cell counter (Sysmex XE-2100; TOA Medical Electronics, Japan) was used to measure the levels of RBCs (number/μl), hemoglobin (Hb, g/dl), hematocrit (Hct, %), and mean corpuscular volume (MCV, fl). For the pre-B CFC assay, whole BM cells (2 × 10⁵ cells per dish) were divided into 35 mm dishes with MethoCult^TM M3630 (Stem Cell Technologies, Canada). After 7 days, the number of colonies formed was counted. For colony-forming unit (CFU) assays, whole BM (3 × 10⁴ cells per dish), peripheral blood (1 × 10⁶ cells per dish), and spleen (0.5 × 10⁶ cells per dish) cells were incubated in 35 mm dishes with MethoCult^® GF M3434 (Stem Cell Technologies). After 12 days, the numbers of CFU-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) colonies were counted using standard criteria. For colony forming unit-erythroid (CFU-E) assay, whole BM cells (2 × 10⁵ cells per dish) from mutants and WT littermates were plated into 35 mm dishes with MethoCult^TM M3334 (Stem Cell Technologies). After 14 days of incubation, colonies formed were counted under optic microscopic observation (Carl Zeiss, Germany).