P4543

The animal protocol for this study was approved by the NCI/CCR Animal Care and Use Committee (ASP SOP-001). QGP-1 or BON-1 cells (5 × 10⁶ cells) were injected subcutaneously into both flanks of five- to seven-week-old athymic nude male mice CAnN.Cg-Foxn1nu/Cr, (The Jackson Laboratory). The resulting tumors were further subcutaneously implanted, to create homogenous tumors. Only tumors from generations 1 to 3 were utilized. Tumor size was monitored using a Vernier caliper, with tumor initiation size ranging from 5 to 7 mm. The mice were euthanized if they exhibited impeded movement, if there were signs of breathing difficulty or tumors exceeded 2 cm in diameter at any point in the study. Mice were randomly grouped into control and treatment groups and then treated via intraperitoneal daily injections with either VPA (300 mg/kg; P4543; Sigma-Aldrich) dissolved in water, CI-994 (5 mg, 7.5 mg, and 10 mg/kg; C0621; Sigma-Aldrich) dissolved in 0.1% DMSO, or 0.1% DMSO as control group. All interventions were done during the light cycle, and mice were not fasted before assessments.

Sodium butyrate (B5887, Sigma-Aldrich) or valproic acid (P4543, Sigma-Aldrich) were dissolved in normal fly food medium at the final concentration of 10 mM. Three groups of flies were prepared: those treated with one of the HDAC inhibitors and those without HDAC inhibitor treatment (control flies). Adult flies aged 1 d were transferred to fly vials containing medium with or without a HDAC inhibitor. At the end of the fifth day of treatment, flies were collected, and their antennae were dissected for RNA extraction. All treatments and experiments were performed at room temperature. Two biological replicates with 60 flies/replicate were performed for each condition, with an average of 23 million reads / replicate, and with an average of 92% mapped. Multiplexed libraries were made from total RNA input using the Illumina TruSeq RNA sample preparation kit (v2) and 50 bps paired-end sequencing was done using the HighSeq2000.

Berberine was obtained from the National Institutes for Food and Drug Control (Beijing, China). Valproate (VPA) (valproic acid sodium salt, P4543) and PTZ (P6500) were purchased from Sigma-Aldrich (St. Louis, MO, United States).
For the seizure model group, we essentially followed the method described by Baraban et al. (2005) (link). Briefly, zebrafish larvae at 7 dpf were exposed to a PTZ solution at concentrations of 2, 4, and 6 mM, respectively, for 1 h and then collected for a behavioral experiment or for 2 h and then collected for in situ hybridization and western blotting experiments. Based on the results of the PTZ dose experiment, 4 mM of PTZ was used for the subsequent experiments conducted in the PTZ–seizure-related groups. Each group contained 24 larvae.
For the drug-treated groups, wild-type larvae and injected larvae at 5 dpf were exposed to BBR at concentrations of 25, 50, and 75 μM or to VPA at concentrations of 60, 120, and 240 μM for 2 days (7 dpf), respectively, after being washed three times with the normal rearing solution. Then, the larvae were exposed to 4 mM of PTZ for 1 h and collected for a behavioral experiment, or after 2 h collected for subsequent experiments including whole-mount in situ hybridization and western blotting detections for c-fos and stx1b transcription and protein levels.

We incubated tadpoles with VPA (1 mM, Sigma-Aldrich, P4543), a broad class I/II HDACs inhibitor, to block histone deacetylase activity. If not stated otherwise, tadpoles were reared in Steinberg’s solution for 48 h.

Stock solutions of pentylentetrazole (PTZ; P6500, Sigma-Aldrich), valproic acid (VPA, P4543, Sigma-Aldrich), tricaine (A5040, Sigma-Aldrich), kynurenic acid (KYN, ab120256, Abcam), tetrodotoxin (TTX) and Baclofen (BAC) were prepared by dissolving in milliQ water (at 100 mM concentration for PTZ, VPA and KYN; 4 g/L for tricaine; 2 mM for TTX; 10 mM for Baclofen;). The final concentrations used in the experiments were obtained by diluting each stock in fish water. D-tubocurarine (D-TC; 93750, Sigma-Aldrich) was dissolved at 2 mM concentration directly in fish water and used by pre-incubating the larvae for 10 min before measurements.

Adult C57 mice and FVB129 mice were bought from the animal center of the Fourth Military Medical University. FMR1^−/− mice were bought from Jax laboratory (stock No.010504). All animal experiments were carried out under protocols approved by the Animal Care and Use Committee of Fourth Military Medical University and according to “Policies on the Use of Animals and Humans in Neuroscience Research,” revised and approved by the Society for Neuroscience.
VPA-treated mice were obtained by breeding the progenies of pregnant mice exposed to VPA at E12.5 (i.p. injection, 500 mg/kg, P4543, Sigma).

The human embryonic stem cell (hESC) line H8 (RRID: CVCL_9389) was obtained from Prof. Wei Jiang (Wuhan University). Cells were maintained in mTeSR1 medium (Cat. 85850, Stem Cells) with feeder cell free. Neuron induction was conducted according to the reported protocol with minor modification. Briefly, hESCs were digested using accutase (Cat. 07920, Stem Cells) and cultured in suspension using a neural induction medium (NIM, Cat.08582, Stem Cells) till embryonic body (EB) formation. EBs were transferred to 6‐well plates until appearance of rosettes. Rosettes were reseeded and cultured with neurobasal medium containing 2% B27, 100 nmol/L brain‐derived neurotrophic factor (Cat. 450‐02, PeproTech) and 100 nmol/L glial‐derived neurotrophic factor (Cat. 450‐10, PeproTech) for neuronal differentiation. VPA (1 mM, P4543, Sigma) was added at 3 days after rosette formation.