FIG. 2.

Inhibition of endogenous Akt by hairpin siRNAs reduces neuron generation by ngn3. (A) P19 cells were transfected with expression vectors for ngn3, nlsGFP, and U6-driven hairpin siRNAs against Akt1 (Akt1 HP7), Akt2 (Akt2 HP5), both Akt1 and Akt2, or a control, XASH3 (XASH3 HP). (B) Cells were transfected as described for panel A, but the caspase inhibitor z-VAD-FMK was added to cells at the time of transfection. Cells were fixed 4 days after transfection, and a neuronal β-tubulin was detected by indirect immunofluorescence with the antibody TuJ1. GFP was detected by epifluorescence. (C and D) The average number of GFP-positive cells and TuJ1-positive cells per field of view in hairpin-transfected cells without (C) or with (D) caspase inhibitor was determined. Averages are derived from counting the number of cells from three fields of view per transfection, from three independent transfections (standard deviations are indicated).