P akt

For immunohistochemical staining, 4-μm-thick sections received antigen retrieval and endogenous peroxidase activity blocking. Then, the samples were incubated in goat serum for 30 min at room temperature to block the nonspecific antigen, followed by incubating in primary antibodies against K10 (1:200; Santa Cruz), K14 (1:200; Santa Cruz), and p-AKT (1:250; Affinity Biosciences) overnight at 4 ℃. Finally, the immune reactivities of the sections were determined using the HRP-streptavidin detection system (ZSGB-bio, Beijing, China). Images from above-mentioned sections were acquired with a BX63 fluorescence microscope (Olympus).
For immunofluorescence staining, 4-μm-thick sections received antigen retrieval and permeabilizing with 0.5% Triton X-100. After blocking with goat serum (Solarbio), primary antibodies against Ki67 (1:250; Affinity Biosciences), Cytokeratin (Pan) (1:200; Huabio, Hangzhou, China), E-cad (1:200; Affinity Biosciences), Vim (1:500; Abcam), and N-cad (1:200; Santa Cruz) were loaded and kept overnight at 4 ℃. Then, Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Abcam) were loaded at room temperature for 2 h. The nuclei were stained with DAPI (Beyotime), with the images visualized via the LSM-980 confocal microscope (Carl Zeiss).

Rat hippocampal tissues were washed 2–3 times with pre-chilled PBS, cut into small pieces and homogenized by adding ten times the volume of tissue in RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Then the supernatant was collected by centrifugation and homogenization (12,000 rpm, 4°C, 10 min), which is the total protein of rat hippocampal tissue. The BCA protein assay kit (AS1086, ASPEN, Wuhan, China) was used for the quantification of total proteins. Samples were separated by SDS PAGE and electro-transferred onto PVDF (0.45 um) membranes (Servicebio,G6015-0.45, Wuhan, China) after activation with methanol. The membranes were then incubated overnight at 4°C with specific primary antibodies:CB1 (Servicebio,GB111214), PI3K(BIOSS,BSM-33219M), P-PI3K (BIOSS,bs5570R), AKT (Servicebio,GB11689), P-AKT (Affinity,AF0908), STAT3 (Servicebio,GB11176), P-STAT3 (RuiyingBiological, RLP0250), Bcl2 (Servicebio, GB113375), BAX (Servicebio, GB11690),ACTIN(Servicebio, GB12001). The membranes were washed three times with TBST for 10 min each. After washing, the membranes were incubated with HRP anti-conjugated secondary antibody (Servicebio, GB23303) for 1 h. The membranes were washed in the same manner as described above. Membranes were scanned using the Fluor Chem FC3 system (Protein Simple, United States).

Whole-cell protein was extracted with the RIPA lysis buffer containing protease inhibitor (Beyotime) and protein phosphatase inhibitor (Beyotime) on ice. Equal amounts of protein (30 μg) were loaded into 10% (w/v) SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, St. Louis, MO, USA), with the membrane then incubated with primary antibodies at 4 °C overnight, followed by three series of TBST washing and 2 h of HRP-conjugated secondary antibodies incubation at room temperature. The proteins were visualized through chemiluminescence and imaged on a Gelview 6000 Pro (BLT Co., Ltd, Guangzhou, China). The protein bands were quantified by densitometry analysis using Image J software.
The primary antibodies used in this study included antibodies against GAPDH (1:1000; Affinity Biosciences), Vim (1:1000; Abcam), E-cad (1:1000; Affinity Biosciences), N-cad (1:1000; Santa Cruz, Cincinnati, OH, USA), PI3K (1:1000; Santa Cruz), AKT (1:1000; Santa Cruz), p-PI3K (1:1000; Affinity Biosciences), p-AKT (1:1000; Affinity Biosciences), and PTHR1 (1:1000; Affinity Biosciences).

The cell plate was permeabilized for 15 min, followed by adding 3% bovine serum albumin (BSA) solution (Solarbio) for blocking at room temperature for 20 min. The BSA was removed from the samples, following which the AKT (1:200; CST), p‐AKT (1:200; Affinity), VEGF (1:200; Affinity), bFGF (1:200; Affinity), and PI3K (1:100; Abcam) antibodies were introduced and incubated at a temperature of 4°C in a humidified environment. All slices were then treated with a secondary antibody (1:500) and incubated in the dark at 37°C for 30 min. The cells were subjected to restaining with 4′,6‐diamidino‐2‐phenylindole for a duration of 5 min in the absence of light. Observation of the cells and collection of images was carried out using a fluorescence microscope (Evident).

Total protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a 0.22 μm polyvinylidene difluoride membrane (Millipore, Billerica, MA). They were then incubated with specific antibodies according to the manufacturer’s protocol. The GAPDH antibody was used as the control. The primary antibodies were as follows: FOXO3a (Abways, CY5079, Shanghai, P.R. China); p-FOXO3a (Abways, CY5562, Shanghai, P.R. China); PI3K (Abways, CY5355, Shanghai, P.R. China); p-PI3K (Abways, CY6427, Shanghai, P.R. China); GAPDH (Abways, AB0037, Shanghai, P.R. China); AKT (Affinity Biosciences, AF6261, USA); p-AKT (Affinity Biosciences, AF016, USA); NF-κB (Affinity Biosciences, AF5006, USA); p-NF-κB (Affinity Biosciences, AF2006, USA); BCL-6 (Affinity Biosciences, DF2903, USA); c-Myb (Affinity Biosciences, AF6136, USA); p53 (Affinity Biosciences, AF0879, USA); cylinG2 (Affinity Biosciences, DF2284, USA); cyclin B1 (Affinity Biosciences, AF6188, USA).