Sox6 Up-Regulation by Macrophage Migration Inhibitory Factor Promotes Survival and Maintenance of Mouse Neural Stem/Progenitor Cells
Figure 3
Sox6 increases survival and/or self-renewal ability of NSPCs.
(A) In the primary neurosphere formation assay, single dissociated cells of neurospheres generated from E14.5 GE were plated onto a 96-well plate and infected with retroviruses encoding GFP (Ctrl) or Sox6 and GFP (Sox6). The cells were cultured in the presence of both EGF and FGF2. In the secondary neurosphere assay, neurospheres infected with a Sox6-expressing retrovirus or control for 5DIV were sorted as GFP-positive cells onto a 96-well plate and cultured in the presence of both EGF and FGF2. (B) Sox6 targeting using retroviral shRNA significantly attenuated the efficiency of both primary and secondary neurosphere formation. In the primary neurosphere assay, single dissociated neurospheres were infected with a retrovirus expressing either Luc-shRNA (shLuc) or Sox6-shRNA (shSox6). In the secondary neurosphere assay, neurospheres infected with a retrovirus expressing either Luc-shRNA or Sox6-shRNA for 5 DIV were dissociated into single cells and plated onto a 96-well plate for the culture in the presence of EGF and FGF2. (C) Schematic representation of the self-inactivating lentivirus vector expressing the Venus reporter gene under the control of the SOX6 promoter. Flow cytometric analysis of neurospheres infected with lentivirus expressing the Venus reporter regulated by a SOX6 promoter for 5 days. (D) Venus-positive cells (II) and Venus-negative cells (I) were sorted onto a 96-well plate and cultured in the presence of EGF and FGF2, and the number of secondary neurospheres was quantified. All neurosphere formation assays were performed at a low cell density (1 cell/µl). Data are representative of three independent experiments. Error bars indicate S.D. values. **P<0.01 versus control; Student’s t-test. (E) The viability of cells dissociated from neurospheres was assessed 5 days after infection with either a control retrovirus or a retrovirus expressing Sox6 using a CellTiter Glo Luminescent Cell Viability Kit. (F) Sox6 targeting via retroviral shRNA significantly reduced NSPC growth compared to Luc-shRNA, as assessed using Cell Titer-Glo Assay Kit 4 days of post-infection. (G) Sox6 targeting via retroviral shRNA led to an increase in caspase 3/7 activity in NSPCs 4 days after infection. Data show a representative data from three independent experiments Error bars indicate S.D. values; *P<0.05, **P<0.01 versus control; Student’s t-test.