FIGURE 6.

Western blot and immunofluorescence analysis of PC12 cells that overexpress Reg-1α and of untransfected neighboring cells. A, PC12 cells were transfected with empty pCMV6-XL5 (lanes 1 and 2) or peGFP (lanes 3 and 4) vector or Reg-1α-GFP (lanes 5 and 6) (see “Materials and Methods”). Cell lysates were collected 48 h after transfection and immunoblotted with the anti-GFP antibody to determine the transfection efficacy. Molecular weight markers (MW) are indicated on the left. B, GFP immunostaining shows that PC12 cells that overexpress Reg-1α-GFP have longer neurites (panel c, arrows) than GFP-transfected cells (panel a, arrow). Panels b and d show the concentric circles around transfected cells (dotted arrows) every 100 μm (0–100 and 100–200 μm). Scale bars, 10 μm. C, quantification of the percentage of undifferentiated (white) and differentiated (i.e. neurite length is twice that of the cell body) (black) cells shows an increase of the number of PC12 cells considered as differentiated when they overexpress Reg-1α-GFP and also of the number of their neighboring cells considered as differentiated in comparison with control, peGFP-transfected cells. Asterisks indicate significant differences from controls following Student's t test analysis (**, p < 0.01). D, quantification of the percentage of undifferentiated (white) and differentiated (i.e. neurite length is twice that of the cell body) (black) cells shows that overexpression of the mutant Reg-1α ΔPS-pIRESeGFP, which lacks the signal peptide, has no effect on the differentiation of transfected PC12 cells and of neighboring cells. ns, no significant differences from controls (Student's t test).