Opti mem 1 media

The Hep1‐6 cells (5 × 10⁵ per well) were seeded into six‑well plates. On reaching 80% confluence, the cells were maintained in serum‑free Opti‑MEM I medium (Thermo Fisher Scientific, Inc.). They were then transfected with 2.5 µg of expression vector pCMV‑HDAC1 (Cat: MG53562‐CF, Sino Biological, Beijing, China) containing mouse HDAC1 cDNA and 5 µL Lipofectamine 3000 reagent (Thermo Fisher Scientific, Inc.) premixed with serum‑free Opti‑MEM I media. The transfected cells were incubated in a CO₂ incubator at 37 °C for 4 h, after which the medium was replaced with standard DMEM containing 10% FBS. Following transfection for 24 h, the stably transfected cells were administered with sodium butyrate for another 24 h.

Heterologous African green monkey kidney fibroblast (COS-7) cells and human embryonic kidney 293 (HEK293) cells were cultured in Opti-MEM I media containing 5% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific). Human fibroblasts were grown in DMEM media (FG0445; Baria) containing 10% FBS and 1% (w/v) gentamycin (Thermo Fisher Scientific). All animal experiments were performed in accordance with institutional ethics guidelines and regulations for ensuring animal welfare. The experiments involving human fibroblasts were performed in accordance with the Declaration of Helsinki of the World Medical Association and was approved by each institute’s respective Medical Ethics Committee. Informed and written consent was obtained from the parents of the patients who provided fibroblasts. All cell lines were transfected with DNA vectors using Lipofectamine 2000 (Thermo Fisher Scientific; Kaniakova et al., 2012a (link)). After transfection, the HEK293 cells used for electrophysiology were dissociated using trypsin. The cells used for microscopy and biochemistry were cultured without the trypsinization step. Experiments were performed within 24–48 h of transfection.