Figure 7.

Apoptosis and autophagy are activated in a developmentally regulated manner in MCL-1-null animals. (A) Brains from E12.5 Foxg1 Cre MCL-1 animals were analysed by western blot for the presence of the caspase-cleaved fragment of the caspase-3 substrate p130^CAS and the autophagic markers LC3 and p62. (B) Autophagy in Foxg1 Cre MCL-1^Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top and middle panels) objectives. Scale bars=50 μm. (C) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15.5 wild-type embryos. Data are expressed as the percentage of LC3 vesicles that are LAMP1-positive±s.d. About 100 LC3 vesicles were counted per section, with four sections per embryo. VZ, ventricular zone. (D) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis (BIM, Puma) and autophagy (ATG7, ATG5-ATG12) related proteins as well as Tuj-1 (neuronal marker), Nestin (neural progenitor marker) and HSC70 (loading control). (E) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL-1 and actin as a loading control. (F) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130^CAS, LC3 and actin as a loading control. Alternatively, they were analysed by immunofluorescence for cytochrome c release (G) in the presence of 50 μM zVAD-FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3–7 embryos±s.d. ^*P<0.001.