Isolation, cultivation and cryopreservation of primary NSCs
Primary NSCs were removed from cerebral cortex tissue of E14.5 fetal rats on ice and digested mechanically into single cells. The cells were collected by centrifuging at 300 g for 5min and resuspended in complete NSC medium, which consists of DMEM/F12 culture medium (HyClone, SH30023.01B), 1×B27 (Gibco, 17504044), 1×N2 (A1370701), 20 ng/ml EGF (Gibco, PHG0311), 10 ng/ml bFGF (Gibco, 13256-029) and 20 ng/ml heparin sodium (Sigma, H3149). The NSCs were seeded into petri dishes at a density of 1×106/ml and cultured in 37 ℃ in an incubator with 5% CO2 incubator, labelled as the P0. After culture for 4 days, when the NSCs had grown into neurospheres with diameters of approximately 100µm, the cells were digested, collected, resuspended in complete NSC culture medium and labelled as the P1 generation. The P1 generation NSCs were resuspended in cell freezing medium (Cyagen, NX-07031-20) at a density of 5×106/ml, aliquoted into cryopreservation tubes, stored at -80℃ overnight and then kept in liquid nitrogen for long-term storage.
Characterization of primary NSCs and C17.2 cells
P3 generation NSCs were seeded into petri dishes t a density of 1×106/ml and cultured overnight. Then, the cells were either treated with different concentrations of BHB for 1 day or pretreated with 500 ng/ml PTX (List Biological, 181), 10 µM gallein (Topscience, T3040), 20 µM NF023 (Abcam, ab120454) or 100 nM U0126 (Sigma, U120) separately for 1 hour, followed by treatment with 0.02 mM BHB for 2 days. Pictures of neurospheres were taken under a microscope and the diameters of the neurospheres were calculated using Image J.
C17.2 cells were purchased from Shanghai Honsun Biological Technology. The cells were seeded into 96-well plates at a density of 5×104/ml and cultured overnight. The cells were either treated with different concentrations of BHB for 1 day or pretreated with 500 ng/ml PTX, 10 µM gallein, 20 µM NF023 or 100 nM U0126 for 1 hour, followed by treatment with 0.02 mM BHB for 1 day. Then, 10 µl CCK-8 (Topscience, T0005) was added into each well and cells were cultured for another 1 hour. The absorption of the solution at 450nm was measured using a SpectraMax iD5 Multi-Mode Microplate reader.
Cell cycle analysis by flow cytometry
C17.2 cells were treated with different concentrations of BHB for 1 day. After digesting into single cells using 0.25% trypsin, the cells were resuspended in 1 ml of pre-cooled phosphate-buffered saline (PBS), dropwise dispersed into 2.33 ml of pre-cooled ethanol and fixed at -20℃ overnight. The fixed cells were collected, washed and resuspended in 1 ml of PBS, followed by staining with 50 µg/ml PI (Sigma, P4170) and 100 µg/ml RNase A (Sigma, 556746) for 30 min. The stained cells were filtered through a 200-mesh sieve and the cell cycle was analyzed by flow cytometry. ModFit was used to analyze the ratio of cells in each phase.
RT-qPCR
Transcription levels of Gpr41, Gpr109a, Pax6 and Sox2 in P3 generation NSCs and C17.2 cells were detected after pretreatment with 500 ng/ml PTX for 1 hour, followed by treatment with 0.02 mM BHB for 1 hour. The transcription levels of Ccnd1 (encoding cyclin D1), Ccne1 (cyclin E1), Cdk4 and Cdk2 in C17.2 cells were detected after pretreatment with serum-free DMEM culture medium for 12 hours, followed by treatment with 0.02 mM BHB for 3 hours. Total RNA was harvested using the RNA simple Total RNA kit (TIANGEN, DP419) and reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, 4897030001) in a T100 Thermal Cycler (BioRad, 1861096). Quantitative PCR was carried out in a CFX96 Touch Real-Time PCR Detection System (BioRad, 1855195) using Ace qPCR SYBR Green Master Mix (Vazyme, Q111-03). The sequences of primers used for qPCR are shown in Supplementary Table S1.
Western blot analysis
P3 generation NSCs and C17.2 cells were treated with BHB for 15 min, 1 hour and 3 hours, respectively. The levels of phospho-Erk1/2 (p-Erk1/2), phospho-JNK (p-JNK), phospho-p38 (p-p38) and phospho-Akt (p-Akt) were detected by western blotting after treatment with BHB for 15 min. The levels of acetylated of H3K9 (H3K9ac), H3K14 (H3K14ac), H3K27 (H3K27ac) and trimethylated H3K4 (H3K4me3) were detected after treatment with BHB for 1 hour. The expression of GPR41 and GPR109A, as well as cyclin D1, cyclin E1, CDK4, CDK2 and p21, was detected after treatment with BHB for 3 hours.
Total protein harvested from NSCs and C17.2 cells was loaded onto SDS-PAGE gel and electrophoresis was conducted in a Mini-protein electrophoresis tank. Target proteins were transferred to an Immobilon-FL PVDF membrane using a Mini Trans-Blot platform. The membrane was blocked using 5% BSA in TBST, and then incubated with the primary antibody overnight at 4°C, followed by washing with PBS and incubation with the secondary antibody for 1 hour at room temperature. Protein bands were observed in a chemiluminescence apparatus by adding Clarity Western ECL Substrate. Semiquantitative analysis of protein bands was conducted using Quantity One. The following primary antibodies were used for western blotting: GPR41 (Santa Cruz, sc-131166), HM74a (Santa Cruz, sc-134583), phospho-Erk1/2 (CST, 4370s), phospho-JNK (CST, 9255), phospho-p38 (CST, 4511), phospho-Akt (CST, 4060), cyclinD1 (CST, 2978), cyclinE1 (CST, 4129), CDK4 (CST, 12790), CDK2 (CST, 2546), p21 (CST, 2947), H3K4me3 (Abcam, ab213224), H3K9ac (Abcam, ab32129), H3K14ac (Abcam, ab52946), H3K27ac (Abcam, ab177178) and H3 (Abcam, ab1791), GAPDH (Proteintech, 60004-1-Ig). The following secondary antibodies were used for western blotting: Goat Anti-Rabbit IgG (H + L) HRP (Affinity, S0001), Goat Anti-Mouse IgG (H + L) HRP (Affinity, S0002) and Rabbit Anti-Goat IgG (H + L) HRP (Affinity, S0010).
Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR)
C17.2 cells were treated with 0.02 mM BHB for 1 hour. Histone modifications in the promoters of the Ccnd1, Cdk4, Ccne1, Cdk2, Pax6 and Sox2 genes were measured using the SimpleChIP Enzymatic Chromatin IP kit (CST, 9003). DNA harvested by ChIP was quantified by qPCR. The primer sequences of the Pax6 and Sox2 promoters are shown in Supplementary Table S2.
Statistical analysis
Values are presented as the means ± SEM, and statistical analysis was conducted using GraphPad Prism. Student’s t-test was performed to compare pairs of groups, and one-way ANOVA followed by Dunnett’s multiple comparisons test and Tukey’s multiple comparisons test was performed to compare more than two groups. Differences with p-values of less than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001