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Effects of STAP-2 on B-cell recovery
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Figure 6. STAP-2 deficiency attenuates the inhibitory effects of lipopolysaccharide (LPS) in pre-B cells. (A and B) Phosphorylation status of P38/MAPK (pT180/pY182), ERK1/2 (pT202/pY204), and STAT5 (pY649) in B220+ cells from wild-type (WT) or STAP-2 transgenic (Tg) mice, under steady state (A) and 12 hours after sub-lethal irradiation (B), were analyzed by flow cytometry. (C-E) Three days after 1.0 mg /kg of LPS had been administered intraperitoneally, flow cytometric analysis was conducted. Number of B-lineage progenitor subsets (pro-B; B220+ CD43+ CD19+ IgM–, pre-B; B220+ CD43– CD19+ IgM–, immature B; B220+ CD43– CD19+ IgM+) in bone marrow (BM) (C), as well as white blood cells (WBC) and B220+ CD19+ B cells in peripheral blood (D) are shown. BrdU was administered 12 hours before analysis of the cell cycle status in B220+ CD19+ cells (E, left). Cell apoptosis was analyzed by Annexin V and 7AAD in B220+ cells using flow cytometry (E, right). Data represent three independent experiments (n=6 in each). (F-I) Indicated concentration of LPS was added into CFU pre-B (F) or B-progenitor suspension cultures (G-I) using cells derived from STAP-2 knockout (KO), Tg, or WT mice. Recovered colony (F) or cell (G) numbers were counted, and the proportion normalized to cells without LPS stimulation (control) were calculated. (H) Cell apoptosis was analyzed by Annexin V and 7AAD using flow cytometry after 4-day cultures with LPS (1.0 mg/mL). (I) Differentiation to IgM+ immature cells was evaluated via flow cytometry. Results are shown as mean±standard deviation. Statistical significances relative to WT were determined by unpaired two-tailed Mann-Whitney tests: *P<0.05 and **P<0.01.
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