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Effects of STAP-2 on B-cell recovery
eage distribution in donor-derived cells showed that the proportion of B cells was increased in KO donors, com- pared to that in WT donors (Figure 1C). Two months fol- lowing transplantation, B-cell chimerism reached a plateau at which the engraftment and lineage distribution were indistinguishable between KO and WT donors
(Figure 1B). Interestingly, we noticed that the proportion of KO donor-derived B cells one month after transplanta- tion was similar to that at two months, indicating that B- cell reconstitution was completed by one month in KO LSK transplanted mice, while it took two months in WT control.
A
B
C
D
E
Figure 2. STAP-2 overexpression impairs early B lymphopoiesis in bone marrow (BM). BM cells from transgenic mice (Tg) that overexpress human STAP-2 under the control of an Em enhancer and Lck proximal promoter were used to evaluate roles of STAP-2 in early B lymphopoiesis. (A) The expression level of the inserted human STAP-2 was analyzed with real-time polymerase chain reaction (RT-PCR). The transcript levels in the indicated subsets (LSK; Lineage- Sca1+ cKithigh, common lymphoid
progenitor [CLP]; Lin– Sca1+ cKitlow Flk2high IL7Rα+ , pro-B; B220+ CD43+
cell (LN) samples in Tg mice. (B) The percentages of Ter119+ erythroid cells, Gr1+ or Mac1+ myeloid cells, CD19+ B cells, or CD3+ T cells, were determined by flow cytometry, and exact numbers were calculated. (C and E) Without initial separation, the samples were stained for lineage-associated surface markers for flow cyto- metric analysis. Representative gating for B-progenitor subsets is shown in (C) (top right panel). Proportion and number of cells in BM of B220+ CD19– CD11clow Ly6C+
plasmacytoid dendritic cells (pDC) and B220– CD19– CD11c+
CD19+ IgM–, pre-B; B220+ CD43- CD19+ IgM) were normalized to the median of lymph node
conventional dendritic cells (cDC) were analyzed (E). (D) Ki67 and 7AAD staining was used for the analy- cells (left panel). Cell apoptosis was analyzed by Annexin V and 7AAD in B220+ cells using flow cytometry (right panel). Similar results were obtained in three independent experiments. Six to ten mice were used for each experiment. Results are presented as mean±standard deviation. Statistical significances relative to wild-type (WT) control were determined by unpaired two-tailed Mann-Whitney test: *P<0.05; **P<0.01. KO: knockout; CLP: com-
sis of the cell cycle status in B220+ CD19+
mon lymphoid progenitor; HSPC: hematopoietic stem/progenitor cells.
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