Figure 6.

Evi2b knockdown impairs repopulating ability of HSPC. (a) Representative dot plot showing engraftment of donor CD45.2⁺ cells transduced with control (NSC1 and NSC2) or Evi2b-silenced (Sh3 and Sh4) constructs and support CD45.1 spleen cells (upper panels). GFP⁺ cells within the CD45.2⁺ gate are shown in the lower panels. Numbers indicate percentage of cells. Blood analysis was performed 4 weeks after transplantation. (b) Quantification of contribution of control or Evi2b-silenced LKS cells to hematopoietic system of the recipient animals 4 weeks after transplantation. The y axis indicates the percentage of GFP⁺ cells out of CD45.2⁺ gate. Each group contains at least 10 animals. (c) Number of GFP⁺ granulocytes derived from the donor (CD45.2⁺) cells. The Y axis indicates the number of GFP⁺Gr/Mac1⁺ × 10³ granulocytes per 100.000 Ly5.2⁺ cells. Each group contains at least five animals. The values on y axes of (b) and (c) were normalized to the original percentage of GFP⁺ cells measured 2 days after infection. Transplantation experiments were performed three times. (d) Representative contour plots of cell cycle analysis on murine LKS cells transduced with controls NSC1 and NSC2 or Evi2b-downregulating shRNAs Sh3 and Sh4. The x axis indicates Hoechst 33342 staining and the y axis shows Pyronin Y staining. The x and y axes are displayed in linear scale. Numbers indicate the percentage of cells in G₀ (lower left quadrant), G₁ (upper left quadrant), and G₂/S/M phase (upper right quadrant). Right panel indicates quantification of cell cycle analysis in LKS cells transduced with the distinct shRNAs. Black bars indicate percentage of cells in G₀, gray bars indicate percentage of cells in G₁, and white bars the percentage of cells in G₂/S/M. Results are average of three independent experiments