Figure 4

Nolz1 regulates proliferation and self-renewal of NPCs. (A) hNolz over-expression produces a down-regulation of endogenous mouse Nolz1 (mNolz1) at 2 and 5 DIV after transfection with hNolz1-DsRED. Values are expressed as the mean percentage of control transfection (DsRED alone), considered as 100%. (B) Western blot of NPC protein extracts 5 DIV after transfection with DsRED control (Cont.) or hNolz-DsRED (hNolz), showing an increase in Nolz after hNolz transfection. (C) Western blot of NPC protein extracts 2 DIV after transfection of Nolz1 small interfering RNA (siRNA) or control siRNA. The expression of Nolz1 decreases about 82% in the cultures transfected with the Nolz1 siRNA relative to those transfected with the control siRNA. (D,E) Over-expression of hNolz in NPCs reduces the number of bromodeoxyuridine (BrdU)-positive cells, whereas Nolz1 siRNA transfection causes an increase in the number of BrdU-positive cells. Results are expressed as the relative number of BrdU-positive cells, standardized to their respective control, considered 100% (dotted line). (F) Nolz1 over-expression results in a decrease in neurosphere generation, while Nolz1 silencing results in an increase in the number of neurospheres just after transfection (P0, passage 0). When cells are dissociated and plated again (P1, passage 1), no differences were observed in any condition. Results are expressed as the percentage of counted neurospheres with respect to their respective control, considered 100% (dotted line). (G) Cell-cycle exit index was analyzed as the percentage of BrdU+/Ki67- cells with respect to the total number of BrdU-positive cells after a 3-DIV pulse label. hNolz induces a significant increase in the number of cells that leave the cell-cycle with respect to enhanced green fluorescent protein (EGFP)-over-expressing cells. The results in each graph represent the mean ± standard error of the mean from at least three independent samples at each condition. Statistical analysis was performed with the Student's t-test. *P < 0.05, ***P < 0.001 relative to the respective controls.