Figure 6
The sensitivity to induction of the intrinsic pathway of apoptosis correlates with En1 expression. (A-D) Ventral midbrain cultures 24 hours after application of apoptosis-inducing compounds. Charts of EnHT(En1+/-;En2-/-) and En2-/- E12 cultures depicting the number of surviving TH-positive cells (A, B) and cultures of En1-inducible MN9D cells depicting cell survival measured by cell proliferation assay (C, D). Surviving cells were normalized against untreated EnHTcultures (A) or untreated non-induced MN9D cells (C), treated EnHTcultures (B) or treated non-induced MN9D cells (D). Higher Engrailed expression reduced the cell death rate after MPP+, HA14-1 and chelerythrine chloride (CC) treatment, whereas the rate of cell survival after application of the tumor necrosis factor alpha (TNFa), Prima-1 and Apoptosis Activator-2 (AA2) does not correlate with the level of En1 expression. Dox, doxycycline. (E) Western blot analysis of mitochondrial and cytoplasmic protein fractions of MN9D cells 72 hours after En1 induction and 24 hours after MPP+ treatment. (F) Proportion of cytochrome C (Cyt-C) in cytosol is lower in En1-expressing MN9D cells before and after MPP+ treatment. Scale bars: 25 μm. Error bars indicate standard error. 6 ≤ n ≤ 27, *p ≤ 0.001, **p ≤ 0.0001. Cyt, cytosolic; Mit, mitochondrial; ANT, adenine nucleotide transporter; Dox, doxycycline.