Casp3

Total protein was extracted using Neuronal Protein Extraction Reagent (ThermoFisher Scientific), and proteins were separated by electrophoresis on a 12% polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were incubated overnight with the primary antibodies at 4 °C with optimal dilution following the manufacturer’s recommendations, and then incubated with the appropriate secondary antibody at a 1:2000 dilution for 1 h at room temperature. The FOXO3 (Cat No. 2497, Cell Signaling Technology, Danvers, MA, USA), p-FOXO3 (Thr32) (Cat No. 9464S, Cell Signaling Technology), p-AKT (Ser473) (Cat No. 4060, Cell Signaling Technology), BCL2 (Cat No. 7382, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CASP3 (Cat No. 7184, Santa Cruz Biotechnology), PTEN (Cat No. 9188, Cell Signaling Technology), and ACTIN β (Cat No. 4967, Cell Signaling Technology) primary antibodies were used in the Western blot experiments. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Cell Signaling Technology) for 1 h at room temperature. The specific signals were detected with enhanced chemiluminescence (Western Bright ECL HRP substrate; Advansta, Menlo Park, CA, USA) and Lucent Blue X-ray film (Advansta).

The paraffin was removed from the slides, and they were rehydrated. The nonspecific binding sites were blocked by incubating in 2% IgG-free bovine serum albumin (BSA, Sigma) at room temperature. Afterwards, the specimens were incubated with 0.2% Triton X-100 overnight at 4–8°C with primary antibodies: GFAP (1 : 1000, Millipore) to mark astrocytes and caspase-3 (casp-3; 1 : 100, Santa Cruz Biotechnology Inc.) that were determined by anti-rabbit FITC-labelled secondary antibodies (1 : 100, Jackson ImmunoResearch Laboratories Inc.). The slides were counterstained with Vectashield with DAPI (Vector Labs) for nuclear staining.
Photomicrographs were taken using a fluorescence microscope Olympus BX-41 and three consecutive slices of each brain tissue were used to observe CA1 hippocampal neurons at 40x. The number of GFAP- and casp-3-immunoreactive cells was quantified in the CA1 subfield of the Hp. Four fields per slide were analyzed and graphically expressed as an average per group ± SE. The images were taken using a digital camera acopled at BX-41 Olympus microscope and the analysis were did with Image Pro Premier of Media Cybernetics.

Protein was isolated from DAW22‐treated MPNST cell lines using the Qproteome Mammalian Protein Prep Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. Concentrations of protein were determined by Bradford Protein Assay (Bio‐Rad, California, USA) followed by denaturation as described by the manufacturer. Protein was separated on a 12% SDS‐PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Massachusetts, USA). Protein on the membrane was first incubated with indicated primary antibodies at 4°C overnight, followed by corresponding secondary antibodies’ incubation at room temperature for 1 hour. Targeted proteins were detected using a horseradish peroxidase‐conjugated chemiluminescent kit (Millipore). AKT (#2920), phospho‐AKT (#4060), ERK (#4695), phospho‐ERK (#4370), poly(ADP‐ribose) polymerase 1 (PARP) (#9532), Non‐phospho (active) CTNNB1 (#8814), caspase 3 (CASP3), and ACTB (#4970), with the exception for CASP3 that was purchased from Santa Cruz Biotechnology (Texas, USA), remaining primary antibodies were from Cell Signaling Technology (Massachusetts, USA). ACTB was used as the loading control.