Figure 6.
Overexpression of CD19 rescuesthe defect of Mst1 deficiency. WT bone marrow cells were transduced with GFP-tagged retroviral vector only. Mst1 KO bone marrow cells were transduced with GFP-tagged retroviral vector expressing with or without CD19, and then transferred into CD45.1 mice recipients together with CD45.1 WT bone marrow cells. After reconstitution, CD45.2 +GFP+ donor-derived splenic B cells were sorted and incubated with lipid bilayers at 37°C. Cells were fixed, permeabilized, and stained for pY and pBtk and then analyzed using TIRFm. Shown are representative images (A and B) and the average values (± SD) of the B cell contact area (C), the MFI of the BCR (D), and the MFI of the pBtk (E) and pY (F) in the contact zone. Sorted CD45.2 +GFP+ donor-derived splenic B cells were incubated with AF546–mB-Fab′–anti-Ig for 10 minutes at 4°C to label the BCR. Then, the cells were incubated with sAg at 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for pBtk and pY (G and H). The MFI of pBtk (I) and pY (J) and colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). WT or Mst1 KO bone marrow cells were transduced with GFP-tagged retroviral vector expressing with or without CD19 and then transferred into CD45.1 mice recipients, and CD45.2 +GFP+-derived MZ B cells were analyzed by flow cytometry after bone marrow reconstitution (L). The quantification of percentage (M) and number (N) of CD45.2 +GFP+-derived MZ B cells in the spleen of WT and Mst1 KO chimera mice. WT or Mst1 KO bone marrow cells were transduced with GFP-tagged retroviral vector expressing with or without CD19 and then transferred into CD45.1 mice recipients subsequently immunized with OVA; CD45.2 +GFP+-derived GC B cells were analyzed by flow cytometry after bone marrow reconstitution (O). The quantification of percentage and number of CD45.2 +GFP+-derived GC B cells in the spleen of WT and Mst1 KO chimera mice (P-Q). Scale bars, 2.5 μm. *P < .01, **P < .001.

Overexpression of CD19 rescuesthe defect of Mst1 deficiency. WT bone marrow cells were transduced with GFP-tagged retroviral vector only. Mst1 KO bone marrow cells were transduced with GFP-tagged retroviral vector expressing with or without CD19, and then transferred into CD45.1 mice recipients together with CD45.1 WT bone marrow cells. After reconstitution, CD45.2 +GFP+ donor-derived splenic B cells were sorted and incubated with lipid bilayers at 37°C. Cells were fixed, permeabilized, and stained for pY and pBtk and then analyzed using TIRFm. Shown are representative images (A and B) and the average values (± SD) of the B cell contact area (C), the MFI of the BCR (D), and the MFI of the pBtk (E) and pY (F) in the contact zone. Sorted CD45.2 +GFP+ donor-derived splenic B cells were incubated with AF546–mB-Fab′–anti-Ig for 10 minutes at 4°C to label the BCR. Then, the cells were incubated with sAg at 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for pBtk and pY (G and H). The MFI of pBtk (I) and pY (J) and colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). WT or Mst1 KO bone marrow cells were transduced with GFP-tagged retroviral vector expressing with or without CD19 and then transferred into CD45.1 mice recipients, and CD45.2 +GFP+-derived MZ B cells were analyzed by flow cytometry after bone marrow reconstitution (L). The quantification of percentage (M) and number (N) of CD45.2 +GFP+-derived MZ B cells in the spleen of WT and Mst1 KO chimera mice. WT or Mst1 KO bone marrow cells were transduced with GFP-tagged retroviral vector expressing with or without CD19 and then transferred into CD45.1 mice recipients subsequently immunized with OVA; CD45.2 +GFP+-derived GC B cells were analyzed by flow cytometry after bone marrow reconstitution (O). The quantification of percentage and number of CD45.2 +GFP+-derived GC B cells in the spleen of WT and Mst1 KO chimera mice (P-Q). Scale bars, 2.5 μm. *P < .01, **P < .001.

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