Bm1453

Well-grown cell spheres were selected for growing on slides coated with polylysine. After drying at 37°C, the slides were washed with phosphate-buffered saline (PBS) three times. The cells were fixed with paraform for 30 min at room temperature, then washed with PBS a further three times. After blocking with 5% goat serum at 37°C for 30 min, primary monoclonal rabbit anti-human nestin (1:30; BA1289, Boster, Wuhan, China) diluted in 1% BSA was added and the cells were placed in a wet box overnight. Subsequently, the cells were washed with PBS, then secondary monoclonal goat anti-rabbit IgG-fluorescein isothiocyanate antibody (1:50; BA1105, Boster) diluted in 1% BSA was added for incubation for 30 min at 37°C. In addition, a negative control in which PBS was used instead of the primary antibody was performed. The slides were observed using an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan). Immunofluorescent assays for glial fibrillary acidic protein (GFAP) and β-tubulin in the differentiated stem cells were conducted using an identical protocol as that used for nestin, with the exception of the respective antibodies. GFAP and β-tubulin were used to identify glioma cells. They were detected by immunofluorescence using monoclonal GAFP (1:30; BA0056; Boster) antibody diluted in 1% BSA and monoclonal β-tubulin (1:30; BM1453; Boster).

The western blot technique was applied to evaluate the expression of key proteins in PACAP/PAC1 pathway as previously described with slight modifications [62 (link)]. Briefly, protein samples (tissues were collected from mPFC and vHPC, or cells) concentrations were determined using the BCA kit (Pierce Biotechnology, Rockford, IL, USA). Then, equal amounts of protein extract were electrophoresed on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore). After blocking for 1 h at room temperature in 5% skim milk, membranes were incubated in the primary antibodies overnight at 4 ℃. The protein bands were detected with primary rabbit polyclonal antibodies: anti-PAC1(ab183103, Abcam, Cambridge, UK; 1:200 dilution), anti-PKA (4782, CST, MA, USA; 1:1000 dilution), anti-p-CREB (9196, CST MA, USA; 1:1000 dilution), anti-CREB (9197, CST, MA, USA; 1:1000 dilution), anti-BDNF (AB1534S, Millipore, St. Louis, UK; 1:1000 dilution), anti-β-Tubulin (BM1453, BOSTER, Wuhan, China; 1:10000 dilution), and secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig-HRP (ab6721, Abcam, Cambridge, UK; 1:200 dilution). Blots were developed by chemiluminescence and quantitated using ImageJ software (NIH, Bethesda, MA, USA).

Angiotensin II, chloroquine (CQ), Bafilomycin A1(Baf A1) and 3-methyladenine (3-MA) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Primary antibodies for detecting Sirt3 (rabbit monoclonal) (D22A3) [5490], FoxO1 (rabbit monoclonal) (C29H4) [2880], LC3 (rabbit polyclonal) [2775], LC3 (rabbit polyclonal) [3868], Beclin-1 (rabbit monoclonal) (D40C5) [3495] were purchased from Cell signalling Technology (CST, UK). Primary antibodies against ac-FoxO1 (rabbit polyclonal) (FKHR D19) [sc49437], MuRF1 (mouse monoclonal) [sc398608], MAFBx (mouse monoclonal) (sc166806) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Primary antibodies against p62 (mouse monoclonal) [ab56416] was obtained from Abcam. Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.