Fig. 4. RNA interference on primary cell cultures derived from En2^–/– ventral midbrain. (A) Numerical chart of RNA interference normalised against mock-transfected control. Only the transfection with En1-specific siRNA oligo duplexes reduced the numbers of TH-positive cells after 96 hours. n=10 independent experiments for each transfection (Student’s t-test, P=0.001 for mock control versus En1 transfection). Error bars indicate s.d. (B) Time course for the loss of mDA neurons after transfection with En1 oligos normalised against mock-transfected controls. Twelve hours after transfection, there were no changes in the numbers of TH-positive neurons; however, a significant proportion of En1-negative cells were present. Number of TH-positive cells gradually decreased until 96 hours when the amount of DA neurons stabilised. Maximum amount of TH-positive and En1-negative cells was detectable at 48 hours. (C-F) Immunohistochemistry on En1 transfected cultures after 48 hours. The transfected TH-positive cells (arrow) are round, have no processes, are En1 negative (red) and possess a fragmented, pyknotic nucleus (DAPI). (C'-F') The mock-transfected control cells (double arrowhead) maintained their neurites and are En1 positive. (F,F') Magnification of DAPI staining in E,E'. (G-J) Confocal image of En1 transfected culture. Three TH-positive neurons are present in this field; one (arrow) has a rounded cell body, is En1 negative and stained positive for activated caspase 3 (blue). Two others (double arrowheads) have spindle shaped cell bodies with attached neurites, are En1 positive and caspase 3 negative. (K-M) Forty-eight hours after transfection with Pbx1-specific RNA duplexes, Pbx1 expression is silenced in some mDA neurons (arrow) but is preserved in the majority (double arrowhead). Regardless of Pbx1 expression, the cells maintained their processes and the shapes of their cell bodies. (N) The degree of silencing 48 hours after transfection with Pbx1- and En1-specific RNA oligos. The same proportion of TH-positive cells, 12%, is negative for Pbx1 and En1. However, the number of TH-positive cells is reduced only after En1 transfection (increase in number after Pbx1 transfection is not statistically significant). Scale bar: 20 µm in C-E,G-M; 10 µm in F.